Prefusion rsv f proteins and their use

ABSTRACT

Disclosed are immunogens including a recombinant RSV F protein stabilized in a prefusion conformation. Also disclosed are nucleic acids encoding the immunogens and methods of producing the immunogens. Methods for generating an immune response in a subject are also disclosed. In some embodiments, the method is a method for treating or preventing a RSV infection in a subject by administering a therapeutically effective amount of the immunogen to the subject.

RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.61/780,910, filed Mar. 13, 2013, and U.S. Provisional Application No.61/798,389, filed Mar. 15, 2013, each of which is incorporated byreference in its entirety.

FIELD

This disclosure relates to polypeptides, compositions, and methods oftheir use, for elicitation and detection of an immune response torespiratory syncytial virus (RSV).

BACKGROUND

Respiratory syncytial virus (RSV) is an enveloped non-segmentednegative-strand RNA virus in the family Paramyxoviridae, genusPneumovirus. It is the most common cause of bronchiolitis and pneumoniaamong children in their first year of life. RSV also causes repeatedinfections including severe lower respiratory tract disease, which mayoccur at any age, especially among the elderly or those with compromisedcardiac, pulmonary, or immune systems. Passive immunization currently isused to prevent severe illness caused by RSV infection, especially ininfants with prematurity, bronchopulmonary dysplasia, or congenitalheart disease. Current treatment includes administration of aRSV-neutralizing antibody, Palivizumab (SYNAGIS®; MedImmune, Inc.),which binds a 24-amino acid, linear, conformational epitope on the RSVFusion (F) protein.

In nature, the RSV F protein is initially expressed as a singlepolypeptide precursor, designated F₀. F₀ trimerizes in the endoplasmicreticulum and is processed by a cellular furin-like protease at twoconserved sites, generating, F₁, F₂ and Pep27 polypeptides. The Pep27polypeptide is excised and does not form part of the mature F protein.The F₂ polypeptide originates from the N-terminal portion of the F₀precursor and links to the F₁ polypeptide via two disulfide bonds. TheF₁ polypeptide originates from the C-terminal portion of the F₀precursor and anchors the mature F protein in the membrane via atransmembrane domain, which is linked to an ˜24 amino acid cytoplasmictail. Three protomers of the F₂-F₁ heterodimer assemble to form a matureF protein, which adopts a metastable prefusion conformation that istriggered to undergo a conformational change that fuses the viral andtarget-cell membranes. Due to its obligatory role in RSV entry, the RSVF protein is the target of neutralizing antibodies and the subject ofvaccine development; however, like other RSV antigens, prior efforts todevelop an RSV F protein-based vaccine have proven unsuccessful.

Prior to the work disclosed herein, a homogeneous preparation of solubleprefusion RSV F protein was unavailable, precluding determination of theprefusion F structure and identification of novel F-specific antigenicsites.

SUMMARY

As described herein, the three-dimensional structure of RSV F protein inits pre-fusion conformation was elucidated. The disclosure reveals forthe first time the prefusion conformation of RSV F, which includes aunique antigenic site (“antigenic site Ø”) at its membrane distal apex.Using the three-dimensional structure of prefusion F as a guide,stabilized forms of prefusion F (“PreF” antigens) were engineered andconstructed and used to generate RSV neutralizing immune responses manyfold greater than that achieved with prior RSV F protein-basedimmunogens.

Disclosed herein are isolated recombinant RSV F proteins that arestabilized in a prefusion conformation, as well as nucleic acidmolecules encoding the recombinant RSV F proteins, which are useful, forexample, to induce an immune response to RSV in a subject. In severalembodiments, the recombinant RSV F protein can be stabilized in aprefusion conformation that can specifically bind to a prefusionspecific antibody, such as a D25 or an AM22 antibody. In someembodiments, the recombinant RSV F protein comprises an antigenic site Øcomprising residues 62-69 and 196-209 of SEQ ID NO: 370, thatspecifically binds the D25 antibody or the AM22 antibody, or both. ThePreF antigens can be used, for example, as both potential vaccines forRSV and as diagnostic molecules. In some embodiments, the recombinantRSV F proteins can be used to detect and quantify target antibodies in apolyclonal serum response.

Elucidation of the PreF antigens was accomplished by achieving, for thefirst time, the crystallization and three-dimensional structuredetermination of the RSV F protein in its prefusion conformation. RSV Fprotein specific antibodies were identified that neutralize RSV, butthat do not bind to a RSV F protein construct stabilized in thepostfusion conformation, and the structure of RSV F protein recognizedby these antibodies was determined. A prefusion-specific antigenic sitewas revealed by the structure (antigenic site Ø), which providesatomic-level details that were used to develop the recombinant RSV Fproteins.

In several embodiments, the RSV F protein can be a single chain RSV Fprotein.

In some embodiments, the recombinant RSV F protein can include an F₁polypeptide and an F₂ polypeptide, wherein the F₁ polypeptide, the F₂polypeptide, or both, include at least one modification (such as anamino acid substitution) that stabilizes the recombinant RSV F proteinin the prefusion conformation. In some embodiments, the modification caninclude an amino acid substitution that introduces a non-naturaldisulfide bond, or the substitution can be a cavity-filling amino acidsubstitution.

In one non-limiting example, the recombinant RSV F protein can includeS155C and S290C substitutions.

In some embodiments, the recombinant RSV F protein can be linked to atrimerization domain, such as a Foldon domain, which can furtherstabilize the recombinant RSV F protein in the prefusion conformation.In additional embodiments, the recombinant RSV F protein can be includedon a protein nanoparticle, such as a ferritin nanoparticle.

Additional embodiments include epitope-scaffold proteins including a RSVF protein prefusion specific epitope (such as antigenic site Ø), whereinthe epitope scaffold protein is specifically bound by a RSV Fprefusion-specific monoclonal antibody, such as a D25 or AM22 antibody.

Methods of generating an immune response in a subject are disclosed, asare methods of treating, inhibiting or preventing a RSV infection in asubject. In such methods a subject, such as a human or bovine subject,can be administered an effective amount of a disclosed PreF antigenand/or a nucleic acid molecule encoding a disclosed PreF antigen. Insome embodiments, the methods include administration of an immunogeniccomposition including an adjuvant selected to elicit a Th1 biased immuneresponse in a subject.

Methods for detecting or isolating an RSV binding antibody in a subjectinfected with RSV are also disclosed.

The foregoing and other objects, features, and advantages of theembodiments will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-1C are a set of graphs and an diagram illustrating RSVneutralization, F glycoprotein recognition, and the crystal structure ofhuman antibody D25 in complex with the prefusion RSV F trimer. Theprefusion conformation of RSV F is metastable, and when expressed in asoluble form readily adopts the postfusion state; a number of potentantibodies, including D25, bind to a newly revealed antigenic site atthe top of the prefusion F glycoprotein. (A) RSV neutralization byantibodies including palivizumab, the FDA-approved prophylactic antibodyto prevent severe RSV disease. (B) Enzyme linked immunosorbant assay(ELISA) measuring antibody binding to postfusion F glycoprotein. (C)D25-RSV F trimer structure in ribbon and molecular surfacerepresentations. One protomer of the F glycoprotein trimer is shown asribbons and colored as a rainbow from blue to red, N-terminus of F₂ toC-terminus of F₁, respectively. Molecular surfaces are shown for theother two F protomers, colored pink and green. The D25 Fab bound to theF protomer shown in ribbons is also displayed in ribbon representation,with heavy chain colored red and light chain colored grey. The other D25Fabs are colored the same, but shown in surface representation.

FIGS. 2A and 2B are a set of diagrams and a sequence aligned with RSVsecondary structure illustrating the structural rearrangement of RSV F.To mediate virus-cell entry, the RSV F glycoprotein transitions from ametastable prefusion conformation to a stable postfusion conformation.(A) Prefusion and postfusion structures. Outer images display prefusion(left) and postfusion (right) trimeric structures, colored the same asin FIG. 1C. A complex glycan, shown as sticks, is modeled at each of thethree N-linked glycosylation sites found in the mature protein. Innerimages display a single RSV F protomer in ribbon representation, coloredas a rainbow from blue to red, N-terminus of F₂ to C-terminus of F₁,respectively. (B) RSV F sequence and secondary structure. Sites ofN-linked glycosylation are highlighted by black triangles, antigenicsites are labeled in red, and downward arrows indicate the position offurin cleavage sites. Secondary structures are shown below the sequence(SEQ ID NO: 370), with cylinders representing α-helices and arrowsrepresenting β-strands. Disordered or missing residues are indicated byan “X”; residues that move over 5 Å between prefusion and postfusionconformations shown with grey shadow.

FIGS. 3A-3C show a set of diagrams and a sequence alignment illustratingthe RSV F interface with D25. Antibody D25 binds a quaternary epitopespanning two protomers at the apex of the prefusion F trimer. (A)Close-up of the interface between D25 and RSV F. Side chains of Fresidues interacting with D25 are labeled and shown as sticks. Oxygenatoms are colored red and nitrogen atoms are colored blue. Hydrogenbonds are depicted as dotted lines. The two images are related by a 90°rotation about the vertical axis. (B) Position and conformation of theD25 epitope on the prefusion and postfusion F molecules. RSV F residuesat the D25 interface are colored red; polarity of α4 and α5_(post)indicated with arrows, with fragment N- and C-termini indicated. (C)Sequence conservation of F residues in regions recognized by D25. Aminoacids in human RSV subtype B (hRSV/B) or in bovine RSV (bRSV) thatdiffer from hRSV/A are colored red. Ectodomain is defined as F residues26-109 and 137-524.

FIGS. 4A-4D are series of graphs and digital images concerning antigenicsite Ø. Highly effective RSV-neutralizing antibodies target a site atthe membrane-distal apex of the prefusion F trimer. (A) The ability ofantibodies to block D25 binding to RSV-infected cells was measured as afunction of antibody concentration. (B) Analysis of RSV F/Fab complexesby negative stain electron microscopy: (Left) Reprojection of a 12 Åslice through the crystal structure of RSV F+D25 Fab filtered to 10 Åresolution and sliced to include the F-trimer cavity. (Middle) Alignedaverage of 263 particles of RSV F+D25 Fab. (Right) Aligned average of550 particles of RSV F+AM22 Fab. Scale bar in middle panel is 50 Å. (C)Fusion inhibition and (D) attachment inhibition activity for antibodiestargeting antigenic site Ø and F-specific antibodies targeting otherantigenic sites. For the attachment-inhibition assay, heparin was usedas a positive control.

FIG. 5 shows a schematic diagram illustrating the methods used toexpress complexes of RSV F and D25. Plasmids expressing RSV F(+) Fd(green circle), the D25 light chain (grey circle), and the D25 heavychain (with or without a stop codon in the hinge region, red circle)were simultaneously transfected into HEK293 cells in suspension.Alternatively, the RSV F(+) Fd plasmid could be transfected, withpurified D25 Fab or IgG added to the cells 3 hours post-transfection.The best yields were obtained by simultaneously expressing F and D25 Fab(˜1.0 mg of purified complex per liter of cells).

FIG. 6 shows a set of ribbon diagrams illustrating the comparison ofD25-bound RSV F to prefusion PIV5 F. Ribbon representation of D25-boundRSV F (+) Fd (left) and PIV5 F-GCNt (right) colored as a rainbow fromblue to red, F₂ N-terminus to F₁ C-terminus, respectively. There isexcellent agreement of secondary structure elements between the twoproteins, despite having only ˜12% sequence identity. One of the moststriking differences is the location of the fusion peptide (N-terminusof F₁ subunit), also shown in FIG. 7. The PIV5 F structure was describedas consisting of three domains: I, II and III (Yin et al., Nature, 439,38 (2006)). Domain III termed the membrane distal lobe, whereas domainsI and II encompass the central barrel and membrane proximal lobe. Thecleaved PIV5 structure shown here was generated from PDB ID: 4GIP (Welchet al., Proc. Natl. Acad. Sci., U.S.A. 109, 16672 (2012)).

FIG. 7 shows a series of diagrams illustrating Type I prefusion viralglycoproteins. Prefusion structures of RSV F, PIV5 F (PDB ID: 4GIP(Welch et al., Proc. Natl. Acad. Sci., U.S.A. 109, 16672 (2012)),influenza HA (PDB ID: 2HMG; Wilson et al., Nature, 289, 366 (1981)) andEbola GP (PDB ID: 3CSY; Lee et al., Nature, 454, 177 (2008)) are shownas molecular surfaces, with each protomer colored differently. On thebottom row, a red sphere is shown for the C-terminal residue of F₂ (RSVand PIV5) or HA₁ (Flu), and a blue sphere is show for the N-terminalresidue of the fusion peptide. The RSV and PIV5 are both paramyxovirusesand their F proteins share ˜12% sequence identity. Although Ebola GP isa type I fusion protein, it lacks a free N-terminal fusion peptide onGP2, and instead contains an internal fusion loop that is commonly seenin type II and type III fusion proteins. Thus, the Ebola GP was omittedfrom the fusion peptide comparison.

FIG. 8 is a set of graphs concerning RSV neutralization by IgG and Fab.D25, AM22 and Motavizumab neutralize RSV equally well as IgG or Fab.Note that the x-axis for the Motavizumab plot is different than theothers.

FIGS. 9A and 9B are a series of diagrams and graphs illustratingproperties of antigenic sites on the RSV F glycoprotein. Only antibodiesdirected to antigenic site Ø bind specifically to the prefusionconformation and have exceptional neutralization potency. (A) For siteØ, an image of a single D25 Fab binding to the prefusion RSV F trimer isshown, along with neutralization curves for AM22 and D25. For site I,arrows point to Pro389, a known escape mutation (Lopez et al., J.Virol., 72, 6922 (1998)). A neutralization curve is shown for antibody131-2a. Like antibody 2F (Magro et al., J. Virol., 84, 7970 (2010)),antibody 131-2a only neutralizes ˜50% of the virus. (B) For antigenicsites II and IV, models of Motavizumab (site II) and 101F (site IV)binding to the prefusion and postfusion (McLellan et al., J. Virol., 85,7788 (2011)) F structures were made using the coordinates ofantibody-peptide structures (McLellan et al., J. Virol., 84, 12236(2010); McLellan et al., Nat. Struct. Mol. Biol., 17, 248 (2010)).

FIG. 10 shows an image of a polyacrylamide gel illustrating expressionof the recombinant RSV F protein construct with S155C and S290C aminoacid substitutions and a Foldon domain linked to the C-terminus of F₁,and a set of diagrams illustrating that the disulfide bond between S155Cand S290C can only form in the prefusion conformation of RSV F protein.

FIG. 11 is a set of graphs showing results from ELISA and gel filtrationassays using the recombinant RSV F protein construct with S155C andS290C amino acid substitutions and a Foldon domain linked to theC-terminus of F₁. The ELISA data indicate that the S155C/S290C constructis specifically bound by RSV F prefusion specific antibodies. The gelfiltration profiles show that the S155C/S290C construct exists solely asa trimer, whereas aggregates and rosettes form in solution with acontrol RSV F construct lacking the S155C/S290C substitutions.

FIG. 12 shows negative-stain electron microscopy images of recombinantRSV F protein construct with S155C and S290C amino acid substitutionsand a Foldon domain linked to the C-terminus of F1. The images below thelarge panel are 2D averages of individual particles. The resultsindicate that the S155C/S290C construct is stabilized in the prefusionconformation.

FIGS. 13-14 show a set of graphs illustrating the neutralizing antibodyresponse of mice administered native RSV (RSV), formalin inactivated RSV(FI-RSV), the recombinant RSV F protein construct with S155C and S290Camino acid substitutions and a Foldon domain linked to the C-terminus ofF₁ (prefusion F), or a RSV F protein construct stabilized in thepostfusion conformation (postfusion RSV). The antibody response at 5weeks (FIG. 13) and 7 weeks (FIG. 14) post-initial immunization isshown.

FIG. 15 shows digital images of the crystals of a soluble recombinantRSV F protein stabilized in a prefusion conformation by S 155C and S290Csubstitutions. Left, standard light images; Right, ultraviolet images,indicative of proteins. The formation of crystals from aqueous bufferedsolutions demonstrates that this protein is substantially homogeneous insolution.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. The Sequence Listing is submitted as an ASCII textfile in the form of the file named “Sequence.txt” (˜1.7 MB), which wascreated on Mar. 12, 2014, and is incorporated by reference herein. Inthe accompanying Sequence Listing:

SEQ ID NOs: 1-128 are the amino acid sequences of native RSV F proteinsfrom RSV type A.

SEQ ID NOs: 129-177 are the amino acid sequences of native RSV Fproteins from RSV type B.

SEQ ID NOs: 178-184 are the amino acid sequences of native RSV Fproteins from bovine RSV.

SEQ ID NOs: 185-350 are the amino acid sequences of recombinant RSV Fproteins.

SEQ ID NO: 351 is the amino acid sequence of a T4 fibritin Foldondomain.

SEQ ID NO: 352 and 355-365 are amino acid sequences of peptide linkers.

SEQ ID NO: 353 is the amino acid sequence of a Helicobacter pyloriferritin protein (GENBANK® Accession No. EJB64322.1, incorporated byreference herein as present in the database on Feb. 28, 2013).

SEQ ID NO: 354 is the amino acid sequence of an encapsulin protein(GENBANK® Accession No. YP_(—)001738186.1, incorporated by referenceherein as present in the database on Feb. 28, 2013).

SEQ ID NOs: 366 and 367 are the V_(H) and V_(L) amino acid sequences ofthe AM22 mAb, respectively.

SEQ ID NO: 368 and 369 are the V_(H) and V_(L) amino acid sequences ofthe D25 mAb, respectively.

SEQ ID NO: 370 is a recombinant RSV F₀ protein variant amino acidsequence of the prototypical A2 strain (GENBANK accession No. P03420,incorporated by reference herein as present in the database on Feb. 28,2012), including P102A, I379V, and M447V substitutions compared to theP03420 sequence.

Structural Coordinates

The atomic coordinates of the crystal structure of RSV F protein boundby D25 Fab are recited in Table 1 of U.S. Provisional Application No.61/780,910, filed Mar. 13, 2013, which is incorporated by referenceherein in its entirety.

DETAILED DESCRIPTION

The RSV F glycoprotein it is a type I fusion protein that facilitatesfusion of viral and cellular membranes (Walsh and Hruska, J. Virol., 47,171 (1983)). After initial synthesis, RSV F adopts a metastableprefusion conformation that stores folding energy, which is releasedduring a structural rearrangement to a highly stable postfusionconformation after contact with host cell membranes. Three antigenicsites (I, II, and IV) on RSV F protein have been found to elicitneutralizing activity (Arbiza et al., J. Gen. Virol., 73, 2225 (1992);Lopez et al., J. Virol., 72, 6922 (1998); López et al., J. Virol., 64,927 (1990)), and all exist on the postfusion form of RSV F protein asdetermined by structural and biophysical studies (McLellan et al., J.Virol., 85, 7788 (2011); Swanson et al., Proc. Natl. Acad. Sci. U.S.A.,108, 9619 (2011)). Absorption of human sera with postfusion RSV F,however, fails to remove the majority of F-specific neutralizingactivity, suggesting that the prefusion form of RSV F harbors novelneutralizing antigenic sites (Magro et al., Proc. Natl. Acad. Sci.U.S.A., 109, 3089 (2012)).

Prior to the work disclosed herein, a homogeneous preparation of solubleprefusion RSV F protein was unavailable, precluding determination of theprefusion F structure and identification of novel F-specific antigenicsites. As described herein, RSV F protein specific antibodies wereidentified that neutralize RSV, but do not specifically bind topostfusion RSV F, and the three-dimensional structure of prefusion F,recognized by these antibodies, was obtained. The results providedherein reveal for the first time the prefusion conformation of RSV F andthe mechanism of neutralization for a category of remarkably potent RSVprefusion F neutralizing antibodies. Using the three-dimensionalstructure of prefusion F as a guide, stabilized forms of prefusion F(“PreF” antigens) were constructed and used to generate RSV neutralizingimmune responses many fold greater than that achieved with prior RSV Fprotein-based immunogens.

I. TERMS

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology canbe found in Benjamin Lewin, Genes VII, published by Oxford UniversityPress, 1999; Kendrew et al. (eds.), The Encyclopedia of MolecularBiology, published by Blackwell Science Ltd., 1994; and Robert A. Meyers(ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995; and other similarreferences.

As used herein, the singular forms “a,” “an,” and “the,” refer to boththe singular as well as plural, unless the context clearly indicatesotherwise. For example, the term “an antigen” includes single or pluralantigens and can be considered equivalent to the phrase “at least oneantigen.” As used herein, the term “comprises” means “includes.” Thus,“comprising an antigen” means “including an antigen” without excludingother elements. It is further to be understood that any and all basesizes or amino acid sizes, and all molecular weight or molecular massvalues, given for nucleic acids or polypeptides are approximate, and areprovided for descriptive purposes, unless otherwise indicated. Althoughmany methods and materials similar or equivalent to those describedherein can be used, particular suitable methods and materials aredescribed below. In case of conflict, the present specification,including explanations of terms, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting. To facilitate review of the various embodiments, thefollowing explanations of terms are provided:

Adjuvant:

A vehicle used to enhance antigenicity. Adjuvants include a suspensionof minerals (alum, aluminum hydroxide, or phosphate) on which antigen isadsorbed; or water-in-oil emulsion, for example, in which antigensolution is emulsified in mineral oil (Freund incomplete adjuvant),sometimes with the inclusion of killed mycobacteria (Freund's completeadjuvant) to further enhance antigenicity (inhibits degradation ofantigen and/or causes influx of macrophages). Immunostimulatoryoligonucleotides (such as those including a CpG motif) can also be usedas adjuvants. Adjuvants include biological molecules (a “biologicaladjuvant”), such as costimulatory molecules. Exemplary adjuvants includeIL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2,OX-40L, 4-1BBL and toll-like receptor (TLR) agonists, such as TLR-9agonists. The person of ordinary skill in the art is familiar withadjuvants (see, e.g., Singh (ed.) Vaccine Adjuvants and DeliverySystems. Wiley-Interscience, 2007). Adjuvants can be used in combinationwith the disclosed PreF antigens.

Administration:

The introduction of a composition into a subject by a chosen route.Administration can be local or systemic. For example, if the chosenroute is intravenous, the composition (such as a composition including adisclosed immunogen) is administered by introducing the composition intoa vein of the subject.

Agent:

Any substance or any combination of substances that is useful forachieving an end or result; for example, a substance or combination ofsubstances useful for inhibiting RSV infection in a subject. Agentsinclude proteins, nucleic acid molecules, compounds, small molecules,organic compounds, inorganic compounds, or other molecules of interest,such as viruses, such as recombinant viruses. An agent can include atherapeutic agent (such as an anti-RSV agent), a diagnostic agent or apharmaceutical agent. In some embodiments, the agent is a polypeptideagent (such as an immunogenic RSV polypeptide), or an anti-viral agent.The skilled artisan will understand that particular agents may be usefulto achieve more than one result.

AM22:

A neutralizing monoclonal antibody that specifically binds to theprefusion conformation of the RSV F protein, but not the post fusionconformation of RSV F protein. AM22 protein and nucleic acid sequencesare known, for example, the heavy and light chain amino acid sequencesof the AM22 antibody are set forth in U.S. Pat. App. Pub. No.2012/0070446, which is incorporated herein in its entirety). Asdescribed in Example 1, AM22 specifically binds to an epitope includingpositions found on the RSV F protein in its prefusion conformation, butnot the post fusion conformation. This epitope is included within RSV Fpositions 62-69 and 196-209, and located at the membrane distal apex ofthe RSV F protein in the prefusion conformation (see, e.g., FIGS. 2B and9A). Prior to this disclosure it was not known that AM22 was specificfor the prefusion conformation. In several embodiments, antibody AM22specifically binds to the PreF antigens disclosed herein.

Amino Acid Substitutions:

The replacement of one amino acid in an antigen with a different aminoacid. In some examples, an amino acid in an antigen is substituted withan amino acid from a homologous protein.

Animal:

A living multi-cellular vertebrate or invertebrate organism, a categorythat includes, for example, mammals. The term mammal includes both humanand non-human mammals. Similarly, the term “subject” includes both humanand veterinary subjects, such as non-human primates. Thus,administration to a subject can include administration to a humansubject. Particular examples of veterinary subjects include domesticatedanimals (such as cats and dogs), livestock (for example, cattle, horses,pigs, sheep, and goats), laboratory animals (for example, mice, rabbits,rats, gerbils, guinea pigs, and non-human primates).

Antibody:

A polypeptide that in nature is substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof, whichspecifically binds and recognizes an analyte (such as an antigen orimmunogen) such as a RSV F protein or antigenic fragment thereof.Immunoglobulin genes include the kappa, lambda, alpha, gamma, delta,epsilon and mu constant region genes, as well as the myriadimmunoglobulin variable region genes.

Antibodies exist, for example as intact immunoglobulins and as a numberof well characterized fragments produced by digestion with variouspeptidases. For instance, Fabs, Fvs, and single-chain Fvs (SCFvs) thatbind to RSV F protein, would be RSV F protein-specific binding agents.This includes intact immunoglobulins and the variants and portions ofthem well known in the art, such as Fab′ fragments, F(ab)′2 fragments,single chain Fv proteins (“scFv”), and disulfide stabilized Fv proteins(“dsFv”). A scFv protein is a fusion protein in which a light chainvariable region of an immunoglobulin and a heavy chain variable regionof an immunoglobulin are bound by a linker, while in dsFvs, the chainshave been mutated to introduce a disulfide bond to stabilize theassociation of the chains. The term also includes genetically engineeredforms such as chimeric antibodies (such as humanized murine antibodies),heteroconjugate antibodies (such as bispecific antibodies). See also,Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford,Ill.); Kuby, J., Immunology, 3^(rd) Ed., W.H. Freeman & Co., New York,1997.

Antibody fragments are defined as follows: (1) Fab, the fragment whichcontains a monovalent antigen-binding fragment of an antibody moleculeproduced by digestion of whole antibody with the enzyme papain to yieldan intact light chain and a portion of one heavy chain; (2) Fab′, thefragment of an antibody molecule obtained by treating whole antibodywith pepsin, followed by reduction, to yield an intact light chain and aportion of the heavy chain; two Fab′ fragments are obtained per antibodymolecule; (3) (Fab′)₂, the fragment of the antibody obtained by treatingwhole antibody with the enzyme pepsin without subsequent reduction; (4)F(ab′)2, a dimer of two Fab′ fragments held together by two disulfidebonds; (5) Fv, a genetically engineered fragment containing the variableregion of the light chain and the variable region of the heavy chainexpressed as two chains; and (6) single chain antibody (“SCA”), agenetically engineered molecule containing the variable region of thelight chain, the variable region of the heavy chain, linked by asuitable polypeptide linker as a genetically fused single chainmolecule. The term “antibody,” as used herein, also includes antibodyfragments either produced by the modification of whole antibodies orthose synthesized de novo using recombinant DNA methodologies.

Typically, a naturally occurring immunoglobulin has heavy (H) chains andlight (L) chains interconnected by disulfide bonds. There are two typesof light chain, lambda (λ) and kappa (κ). There are five main heavychain classes (or isotypes) which determine the functional activity ofan antibody molecule: IgM, IgD, IgG, IgA and IgE. The disclosedantibodies can be class switched.

Each heavy and light chain contains a constant region and a variableregion, (the regions are also known as “domains”). In severalembodiments, the heavy and the light chain variable domains combine tospecifically bind the antigen. In additional embodiments, only the heavychain variable domain is required. For example, naturally occurringcamelid antibodies consisting of a heavy chain only are functional andstable in the absence of light chain (see, e.g., Hamers-Casterman etal., Nature, 363:446-448, 1993; Sheriff et al., Nat. Struct. Biol.,3:733-736, 1996). Light and heavy chain variable domains contain a“framework” region interrupted by three hypervariable regions, alsocalled “complementarity-determining regions” or “CDRs” (see, e.g., Kabatet al., Sequences of Proteins of Immunological Interest, U.S. Departmentof Health and Human Services, 1991). The sequences of the frameworkregions of different light or heavy chains are relatively conservedwithin a species. The framework region of an antibody, that is thecombined framework regions of the constituent light and heavy chains,serves to position and align the CDRs in three-dimensional space.

The CDRs are primarily responsible for binding to an epitope of anantigen. The amino acid sequence boundaries of a given CDR can bereadily determined using any of a number of well-known schemes,including those described by Kabat et al. (“Sequences of Proteins ofImmunological Interest,” 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md., 1991; “Kabat” numbering scheme),Al-Lazikani et al., (JMB 273,927-948, 1997; “Chothia” numbering scheme),and Lefranc, et al. (“IMGT unique numbering for immunoglobulin and Tcell receptor variable domains and Ig superfamily V-like domains,” Dev.Comp. Immunol., 27:55-77, 2003; “IMGT” numbering scheme).

The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3(from the N-terminus to C-terminus), and are also typically identifiedby the chain in which the particular CDR is located. Thus, a V_(H) CDR3is located in the variable domain of the heavy chain of the antibody inwhich it is found, whereas a V_(L) CDR1 is the CDR1 from the variabledomain of the light chain of the antibody in which it is found. Lightchain CDRs are sometimes referred to as CDR L1, CDR L2, and CDR L3.Heavy chain CDRs are sometimes referred to as CDR H1, CDR H2, and CDRH3.

Antigen:

A compound, composition, or substance that can stimulate the productionof antibodies or a T cell response in an animal, including compositionsthat are injected or absorbed into an animal. An antigen reacts with theproducts of specific humoral or cellular immunity, including thoseinduced by heterologous antigens, such as the disclosed recombinant RSVF proteins. “Epitope” or “antigenic determinant” refers to the region ofan antigen to which B and/or T cells respond. In one embodiment, T cellsrespond to the epitope, when the epitope is presented in conjunctionwith an MHC molecule. Epitopes can be formed both from contiguous aminoacids or noncontiguous amino acids juxtaposed by tertiary folding of aprotein. Epitopes formed from contiguous amino acids are typicallyretained on exposure to denaturing solvents whereas epitopes formed bytertiary folding are typically lost on treatment with denaturingsolvents. An epitope typically includes at least 3, and more usually, atleast 5, about 9, or about 8-10 amino acids in a unique spatialconformation. Methods of determining spatial conformation of epitopesinclude, for example, x-ray crystallography and nuclear magneticresonance.

Examples of antigens include, but are not limited to, polypeptides,peptides, lipids, polysaccharides, combinations thereof (such asglycopeptides) and nucleic acids containing antigenic determinants, suchas those recognized by an immune cell. In some examples, antigensinclude peptides derived from a pathogen of interest, such as RSV. Inspecific examples, an antigen is derived from RSV, such as an antigenincluding a RSV F protein in a prefusion conformation.

A “target epitope” is a specific epitope on an antigen that specificallybinds an antibody of interest, such as a monoclonal antibody. In someexamples, a target epitope includes the amino acid residues that contactthe antibody of interest, such that the target epitope can be selectedby the amino acid residues determined to be in contact with the antibodyof interest.

Anti-RSV Agent:

An agent that specifically inhibits RSV from replicating or infectingcells. Non-limiting examples of anti-RSV agents include the monoclonalantibody palivizumab (SYNAGIS®; Medimmune, Inc.) and the small moleculeanti-viral drug ribavirin (manufactured by many sources, e.g., WarrickPharmaceuticals, Inc.).

Atomic Coordinates or Structure Coordinates:

Mathematical coordinates derived from mathematical equations related tothe patterns obtained on diffraction of a monochromatic beam of X-raysby the atoms (scattering centers) such as an antigen, or an antigen incomplex with an antibody. In some examples that antigen can be RSV Fprotein (for example stabilized in a prefusion conformation by bindingto a prefusion-specific antibody, or by introduction of stabilizingmodifications) in a crystal. The diffraction data are used to calculatean electron density map of the repeating unit of the crystal. Theelectron density maps are used to establish the positions of theindividual atoms within the unit cell of the crystal. In one example,the term “structure coordinates” refers to Cartesian coordinates derivedfrom mathematical equations related to the patterns obtained ondiffraction of a monochromatic beam of X-rays, such as by the atoms of aRSV F protein in crystal form.

Those of ordinary skill in the art understand that a set of structurecoordinates determined by X-ray crystallography is not without standarderror. For the purpose of this disclosure, any set of structurecoordinates that have a root mean square deviation of protein backboneatoms (N, Cα, C and O) of less than about 1.0 Angstroms whensuperimposed, such as about 0.75, or about 0.5, or about 0.25 Angstroms,using backbone atoms, shall (in the absence of an explicit statement tothe contrary) be considered identical.

Cavity-Filling Amino Acid Substitution:

An amino acid substitution that fills a cavity within the protein coreof the RSV F protein, for example a cavity present in a protomer of theRSV F protein, or a cavity between protomers of the RSV F protein.Cavities are essentially voids within a folded protein where amino acidsor amino acid side chains are not present. In several embodiments, acavity filling amino acid substitution is introduced to fill a cavity inthe RSV F protein core present in the RSV F protein prefusionconformation that collapse (e.g., have reduced volume) after transitionto the postfusion conformation.

Contacting:

Placement in direct physical association; includes both in solid andliquid form. Contacting includes contact between one molecule andanother molecule, for example the amino acid on the surface of onepolypeptide, such as an antigen, that contact another polypeptide, suchas an antibody. Contacting also includes administration, such asadministration of a disclosed antigen to a subject by a chosen route.

Control:

A reference standard. In some embodiments, the control is a negativecontrol sample obtained from a healthy patient. In other embodiments,the control is a positive control sample obtained from a patientdiagnosed with RSV infection. In still other embodiments, the control isa historical control or standard reference value or range of values(such as a previously tested control sample, such as a group of RSVpatients with known prognosis or outcome, or group of samples thatrepresent baseline or normal values).

A difference between a test sample and a control can be an increase orconversely a decrease. The difference can be a qualitative difference ora quantitative difference, for example a statistically significantdifference. In some examples, a difference is an increase or decrease,relative to a control, of at least about 5%, such as at least about 10%,at least about 20%, at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, at least about 100%, at least about 150%, at leastabout 200%, at least about 250%, at least about 300%, at least about350%, at least about 400%, at least about 500%, or greater than 500%.

D25:

A neutralizing monoclonal antibody that specifically binds to theprefusion conformation of the RSV F protein, but not the post fusionconformation of RSV F protein. D25 protein and nucleic acid sequencesare known, for example, the heavy and light chain amino acid sequencesof the D25 antibody are set forth in U.S. Pat. App. Pub. No.2010/0239593, which is incorporated herein in its entirety; see also,Kwakkenbos et al., Nat. Med., 16:123-128, 2009). As described in Example1, D25 specifically binds to a quaternary epitope found on the RSV Fprotein in its prefusion conformation, but not the post fusionconformation. This epitope is included within RSV F positions 62-69 and196-209, and located at the membrane distal apex of the RSV F protein inthe prefusion conformation (see, e.g., FIGS. 2B and 9A). Prior to thisdisclosure it was not known that D25 was specific for the prefusionconformation of RSV F protein). In several embodiments, antibody D25specifically binds to the PreF antigens disclosed herein.

Degenerate Variant and Conservative Variant:

A polynucleotide encoding a polypeptide or an antibody that includes asequence that is degenerate as a result of the genetic code. Forexample, a polynucleotide encoding a disclosed antigen or an antibodythat specifically binds a disclosed antigen includes a sequence that isdegenerate as a result of the genetic code. There are 20 natural aminoacids, most of which are specified by more than one codon. Therefore,all degenerate nucleotide sequences are included as long as the aminoacid sequence of the antigen or antibody that binds the antigen encodedby the nucleotide sequence is unchanged. Because of the degeneracy ofthe genetic code, a large number of functionally identical nucleic acidsencode any given polypeptide. For instance, the codons CGU, CGC, CGA,CGG, AGA, and AGG all encode the amino acid arginine. Thus, at everyposition where an arginine is specified within a protein encodingsequence, the codon can be altered to any of the corresponding codonsdescribed without altering the encoded protein. Such nucleic acidvariations are “silent variations,” which are one species ofconservative variations. Each nucleic acid sequence herein that encodesa polypeptide also describes every possible silent variation. One ofskill will recognize that each codon in a nucleic acid (except AUG,which is ordinarily the only codon for methionine) can be modified toyield a functionally identical molecule by standard techniques.Accordingly, each “silent variation” of a nucleic acid which encodes apolypeptide is implicit in each described sequence.

One of ordinary skill will recognize that individual substitutions,deletions or additions which alter, add or delete a single amino acid ora small percentage of amino acids (for instance less than 5%, in someembodiments less than 1%) in an encoded sequence are conservativevariations where the alterations result in the substitution of an aminoacid with a chemically similar amino acid.

Conservative amino acid substitutions providing functionally similaramino acids are well known in the art. The following six groups eachcontain amino acids that are conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Not all residue positions within a protein will tolerate an otherwise“conservative” substitution. For instance, if an amino acid residue isessential for a function of the protein, even an otherwise conservativesubstitution may disrupt that activity, for example the specific bindingof an antibody to a target epitope may be disrupted by a conservativemutation in the target epitope.

Epitope:

An antigenic determinant. These are particular chemical groups orpeptide sequences on a molecule that are antigenic, such that theyelicit a specific immune response, for example, an epitope is the regionof an antigen to which B and/or T cells respond. An antibody binds aparticular antigenic epitope, such as an epitope of a RSV F protein, forexample, a D25 or AM22 epitope present on the prefusion conformation ofthe RSV F protein.

Epitopes can be formed both from contiguous amino acids or noncontiguousamino acids juxtaposed by tertiary folding of a protein. Epitopes formedfrom contiguous amino acids are typically retained on exposure todenaturing solvents whereas epitopes formed by tertiary folding aretypically lost on treatment with denaturing solvents. An epitopetypically includes at least 3, and more usually, at least 5, about 9, orabout 8-10 amino acids in a unique spatial conformation. Methods ofdetermining spatial conformation of epitopes include, for example, x-raycrystallography and nuclear magnetic resonance. Epitopes can alsoinclude post-translation modification of amino acids, such as N-linkedglycosylation.

Effective Amount:

An amount of agent, such as a PreF antigen or nucleic acid encoding aPreF antigen or other agent that is sufficient to generate a desiredresponse, such as an immune response to RSV F protein, or a reduction orelimination of a sign or symptom of a condition or disease, such as RSVinfection. For instance, this can be the amount necessary to inhibitviral replication or to measurably alter outward symptoms of the viralinfection. In general, this amount will be sufficient to measurablyinhibit virus (for example, RSV) replication or infectivity. Whenadministered to a subject, a dosage will generally be used that willachieve target tissue concentrations (for example, in respiratorytissue) that has been shown to achieve in vitro inhibition of viralreplication. In some examples, an “effective amount” is one that treats(including prophylaxis) one or more symptoms and/or underlying causes ofany of a disorder or disease, for example to treat RSV infection. In oneexample, an effective amount is a therapeutically effective amount. Inone example, an effective amount is an amount that prevents one or moresigns or symptoms of a particular disease or condition from developing,such as one or more signs or symptoms associated with RSV infection.

Expression:

Translation of a nucleic acid into a protein. Proteins may be expressedand remain intracellular, become a component of the cell surfacemembrane, or be secreted into the extracellular matrix or medium.

Expression Control Sequences:

Nucleic acid sequences that regulate the expression of a heterologousnucleic acid sequence to which it is operatively linked Expressioncontrol sequences are operatively linked to a nucleic acid sequence whenthe expression control sequences control and regulate the transcriptionand, as appropriate, translation of the nucleic acid sequence. Thusexpression control sequences can include appropriate promoters,enhancers, transcription terminators, a start codon (ATG) in front of aprotein-encoding gene, splicing signal for introns, maintenance of thecorrect reading frame of that gene to permit proper translation of mRNA,and stop codons. The term “control sequences” is intended to include, ata minimum, components whose presence can influence expression, and canalso include additional components whose presence is advantageous, forexample, leader sequences and fusion partner sequences. Expressioncontrol sequences can include a promoter.

A promoter is a minimal sequence sufficient to direct transcription.Also included are those promoter elements which are sufficient to renderpromoter-dependent gene expression controllable for cell-type specific,tissue-specific, or inducible by external signals or agents; suchelements may be located in the 5′ or 3′ regions of the gene. Bothconstitutive and inducible promoters are included (see for example,Bitter et al., Methods in Enzymology 153:516-544, 1987). For example,when cloning in bacterial systems, inducible promoters such as pL ofbacteriophage lambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) andthe like may be used. In one embodiment, when cloning in mammalian cellsystems, promoters derived from the genome of mammalian cells (such asmetallothionein promoter) or from mammalian viruses (such as theretrovirus long terminal repeat; the adenovirus late promoter; thevaccinia virus 7.5K promoter) can be used. Promoters produced byrecombinant DNA or synthetic techniques may also be used to provide fortranscription of the nucleic acid sequences.

A polynucleotide can be inserted into an expression vector that containsa promoter sequence, which facilitates the efficient transcription ofthe inserted genetic sequence of the host. The expression vectortypically contains an origin of replication, a promoter, as well asspecific nucleic acid sequences that allow phenotypic selection of thetransformed cells.

Ferritin:

A protein that stores iron and releases it in a controlled fashion. Theprotein is produced by almost all living organisms. Ferritin assemblesinto a globular protein complex that in some cases consists of 24protein subunits. In some examples, ferritin is used to form a particlepresenting antigens on its surface, for example an RSV antigen, such asthe disclosed RSV F protein antigens stabilized in a prefusionconformation.

Foldon Domain:

An amino acid sequence that naturally forms a trimeric structure. Insome examples, a Foldon domain can be included in the amino acidsequence of a disclosed RSV F protein antigen stabilized in a prefusionconformation so that the antigen will form a trimer. In one example, aFoldon domain is the T4 Foldon domain set forth as SEQ ID NO: 351.

Glycoprotein (gp):

A protein that contains oligosaccharide chains (glycans) covalentlyattached to polypeptide side-chains. The carbohydrate is attached to theprotein in a cotranslational or posttranslational modification. Thisprocess is known as glycosylation. In proteins that have segmentsextending extracellularly, the extracellular segments are oftenglycosylated. Glycoproteins are often important integral membraneproteins, where they play a role in cell-cell interactions. In someexamples a glycoprotein is an RSV glycoprotein, such as a RSV F proteinantigen stabilized in a prefusion conformation or an immunogenicfragment thereof.

Glycosylation Site:

An amino acid sequence on the surface of a polypeptide, such as aprotein, which accommodates the attachment of a glycan. An N-linkedglycosylation site is triplet sequence of NX(S/T) in which N isasparagine, X is any residues except proline, and (S/T) is a serine orthreonine residue. A glycan is a polysaccharide or oligosaccharide.Glycan may also be used to refer to the carbohydrate portion of aglycoconjugate, such as a glycoprotein, glycolipid, or a proteoglycan.

Homologous Proteins:

Proteins from two or more species that have a similar structure andfunction in the two or more species. For example a RSV F protein fromone species of RSV such as RSV A is a homologous protein to a RSV Fprotein from a related species such as bovine RSV F protein. Homologousproteins share similar protein folding characteristics and can beconsidered structural homologs.

Homologous proteins typically share a high degree of sequenceconservation, such as at least 80%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, or at least 95%, at least 96%, at least97%, at least 98%, or at least 99% sequence conservation, and a highdegree of sequence identity, such as at least 80%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, or at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% sequenceidentity.

Host Cells:

Cells in which a vector can be propagated and its DNA expressed. Thecell may be prokaryotic or eukaryotic. The term also includes anyprogeny of the subject host cell. It is understood that all progeny maynot be identical to the parental cell since there may be mutations thatoccur during replication. However, such progeny are included when theterm “host cell” is used.

Immunogen:

A protein or a portion thereof that is capable of inducing an immuneresponse in a mammal, such as a mammal infected or at risk of infectionwith a pathogen. Administration of an immunogen can lead to protectiveimmunity and/or proactive immunity against a pathogen of interest. Insome examples, an immunogen includes a disclosed PreF antigen.

Immune Response:

A response of a cell of the immune system, such as a B cell, T cell, ormonocyte, to a stimulus. In one embodiment, the response is specific fora particular antigen (an “antigen-specific response”). In oneembodiment, an immune response is a T cell response, such as a CD4+response or a CD8+ response. In another embodiment, the response is a Bcell response, and results in the production of specific antibodies.

A “Th1” biased immune response is characterized by the presence of CD4⁺T helper cells that produce IL-2 and IFN-γ, and thus, by the secretionor presence of IL-2 and IFN-γ. In contrast, a “Th2” biased immuneresponse is characterized by a preponderance of CD4⁺ helper cells thatproduce IL-4, IL-5, and IL-13.

Immunogenic Composition:

A composition comprising an antigen that induces an immune response,such as a measurable CTL response against virus expressing the antigen,or a measurable B cell response (such as production of antibodies)against the antigen. As such, an immunogenic composition includes one ormore antigens (for example, polypeptide antigens) or antigenic epitopes.An immunogenic composition can also include one or more additionalcomponents capable of eliciting or enhancing an immune response, such asan excipient, carrier, and/or adjuvant. In certain instances,immunogenic compositions are administered to elicit an immune responsethat protects the subject against symptoms or conditions induced by apathogen. In some cases, symptoms or disease caused by a pathogen isprevented (or reduced or ameliorated) by inhibiting replication of thepathogen (e.g., RSV) following exposure of the subject to the pathogen.In one example, an “immunogenic composition” includes a recombinant RSVF protein stabilized in a prefusion conformation, that induces ameasurable CTL response against virus expressing RSV F protein, orinduces a measurable B cell response (such as production of antibodies)against a RSV F protein. It further refers to isolated nucleic acidsencoding an antigen, such as a nucleic acid that can be used to expressthe antigen (and thus be used to elicit an immune response against thispolypeptide).

For in vitro use, an immunogenic composition may include an antigen ornucleic acid encoding an antigen. For in vivo use, the immunogeniccomposition will typically include the protein, immunogenic peptide ornucleic acid in pharmaceutically acceptable carriers, and/or otheragents. Any particular peptide, such as a disclosed RSV F proteinstabilized in a prefusion conformation or a nucleic acid encoding adisclosed RSV F protein stabilized in a prefusion conformation, can bereadily tested for its ability to induce a CTL or B cell response byart-recognized assays. Immunogenic compositions can include adjuvants,which are well known to one of skill in the art.

Immunologically Reactive Conditions:

Includes reference to conditions which allow an antibody raised againsta particular epitope to bind to that epitope to a detectably greaterdegree than, and/or to the substantial exclusion of, binding tosubstantially all other epitopes. Immunologically reactive conditionsare dependent upon the format of the antibody binding reaction andtypically are those utilized in immunoassay protocols or thoseconditions encountered in vivo. The immunologically reactive conditionsemployed in the methods are “physiological conditions” which includereference to conditions (such as temperature, osmolarity, pH) that aretypical inside a living mammal or a mammalian cell. While it isrecognized that some organs are subject to extreme conditions, theintra-organismal and intracellular environment is normally about pH 7(such as from pH 6.0 to pH 8.0, more typically pH 6.5 to 7.5), containswater as the predominant solvent, and exists at a temperature above 0°C. and below 50° C. Osmolarity is within the range that is supportive ofcell viability and proliferation.

Immunological Probe:

A molecule that can be used for selection of antibodies from sera whichare directed against a specific epitope or antigen, including from humanpatient sera. In some examples, the disclosed RSV F proteins stabilizedin a prefusion conformation can be used as immunological probes in bothpositive and negative selection of antibodies specific for RSV F proteinin a prefusion conformation.

Immunogenic Surface:

A surface of a molecule, for example RSV F protein, capable of elicitingan immune response. An immunogenic surface includes the definingfeatures of that surface, for example the three-dimensional shape andthe surface charge. In some examples, an immunogenic surface is definedby the amino acids on the surface of a protein or peptide that are incontact with an antibody, such as a neutralizing antibody, when theprotein and the antibody are bound together. A target epitope includesan immunogenic surface. Immunogenic surface is synonymous with antigenicsurface.

Inhibiting or Treating a Disease:

Inhibiting the full development of a disease or condition, for example,in a subject who is at risk for a disease such as RSV infection.“Treatment” refers to a therapeutic intervention that ameliorates a signor symptom of a disease or pathological condition after it has begun todevelop. The term “ameliorating,” with reference to a disease orpathological condition, refers to any observable beneficial effect ofthe treatment. The beneficial effect can be evidenced, for example, by adelayed onset of clinical symptoms of the disease in a susceptiblesubject, a reduction in severity of some or all clinical symptoms of thedisease, a slower progression of the disease, an improvement in theoverall health or well-being of the subject, or by other parameters wellknown in the art that are specific to the particular disease. A“prophylactic” treatment is a treatment administered to a subject whodoes not exhibit signs of a disease or exhibits only early signs for thepurpose of decreasing the risk of developing pathology.

The term “reduces” is a relative term, such that an agent reduces aresponse or condition if the response or condition is quantitativelydiminished following administration of the agent, or if it is diminishedfollowing administration of the agent, as compared to a reference agent.Similarly, the term “prevents” does not necessarily mean that an agentcompletely eliminates the response or condition, so long as at least onecharacteristic of the response or condition is eliminated. Thus, animmunogenic composition that reduces or prevents an infection or aresponse, such as a pathological response, e.g., vaccine enhanced viraldisease, can, but does not necessarily completely eliminate such aninfection or response, so long as the infection or response ismeasurably diminished, for example, by at least about 50%, such as by atleast about 70%, or about 80%, or even by about 90% of (that is to 10%or less than) the infection or response in the absence of the agent, orin comparison to a reference agent.

Isolated:

An “isolated” biological component (such as a protein, for example adisclosed PreF antigen or nucleic acid encoding such an antigen) hasbeen substantially separated or purified away from other biologicalcomponents in which the component naturally occurs, such as otherchromosomal and extrachromosomal DNA, RNA, and proteins. Proteins,peptides and nucleic acids that have been “isolated” include proteinspurified by standard purification methods. The term also embracesproteins or peptides prepared by recombinant expression in a host cellas well as chemically synthesized proteins, peptides and nucleic acidmolecules. Isolated does not require absolute purity, and can includeprotein, peptide, or nucleic acid molecules that are at least 50%isolated, such as at least 75%, 80%, 90%, 95%, 98%, 99%, or even 99.9%isolated. The PreF antigens disclosed herein (for example, an isolatedrecombinant RSV F protein stabilized in a prefusion conformation)isolated from RSV F proteins in a post-fusion conformation, for example,are at least 80% isolated, at least 90%, 95%, 98%, 99%, or even 99.9%isolated from RSV F proteins in a postfusion conformation. In severalembodiments, the PreF antigen is substantially separated from RSV Fproteins that do not include antigen site Ø and/or are not specificallybound by a prefusion specific monoclonal antibody (such as D25 or AM22),for example, the PreF antigen may be at least 80% isolated, at least90%, 95%, 98%, 99%, or even 99.9% isolated from RSV F proteins that donot include antigen site Ø and/or are not specifically bound by aprefusion specific monoclonal antibody, such as D25 or AM22.

K_(d):

The dissociation constant for a given interaction, such as apolypeptide-ligand interaction or an antibody-antigen interaction. Forexample, for the bimolecular interaction of an antibody (such as D25)and an antigen (such as RSV F protein), it is the concentration of theindividual components of the bimolecular interaction divided by theconcentration of the complex. Methods of determining the Kd of anantibody:antigen interaction are familiar to the person of ordinaryskill in the art.

Label:

A detectable compound or composition that is conjugated directly orindirectly to another molecule to facilitate detection of that molecule.Specific, non-limiting examples of labels include fluorescent tags,enzymatic linkages, and radioactive isotopes. In some examples, adisclosed PreF antigen is labeled with a detectable label. In someexamples, label is attached to a disclosed antigen or nucleic acidencoding such an antigen.

Native Antigen or Native Sequence:

An antigen or sequence that has not been modified by selective mutation,for example, selective mutation to focus the antigenicity of the antigento a target epitope. Native antigen or native sequence are also referredto as wild-type antigen or wild-type sequence.

Nucleic Acid:

A polymer composed of nucleotide units (ribonucleotides,deoxyribonucleotides, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof) linked viaphosphodiester bonds, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof. Thus, the termincludes nucleotide polymers in which the nucleotides and the linkagesbetween them include non-naturally occurring synthetic analogs, such as,for example and without limitation, phosphorothioates, phosphoramidates,methyl phosphonates, chiral-methyl phosphonates, 2-O-methylribonucleotides, peptide-nucleic acids (PNAs), and the like. Suchpolynucleotides can be synthesized, for example, using an automated DNAsynthesizer. The term “oligonucleotide” typically refers to shortpolynucleotides, generally no greater than about 50 nucleotides. It willbe understood that when a nucleotide sequence is represented by a DNAsequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e.,A, U, G, C) in which “U” replaces “T.”

“Nucleotide” includes, but is not limited to, a monomer that includes abase linked to a sugar, such as a pyrimidine, purine or syntheticanalogs thereof, or a base linked to an amino acid, as in a peptidenucleic acid (PNA). A nucleotide is one monomer in a polynucleotide. Anucleotide sequence refers to the sequence of bases in a polynucleotide.

Conventional notation is used herein to describe nucleotide sequences:the left-hand end of a single-stranded nucleotide sequence is the5′-end; the left-hand direction of a double-stranded nucleotide sequenceis referred to as the 5′-direction. The direction of 5′ to 3′ additionof nucleotides to nascent RNA transcripts is referred to as thetranscription direction. The DNA strand having the same sequence as anmRNA is referred to as the “coding strand;” sequences on the DNA strandhaving the same sequence as an mRNA transcribed from that DNA and whichare located 5′ to the 5′-end of the RNA transcript are referred to as“upstream sequences;” sequences on the DNA strand having the samesequence as the RNA and which are 3′ to the 3′ end of the coding RNAtranscript are referred to as “downstream sequences.”

“cDNA” refers to a DNA that is complementary or identical to an mRNA, ineither single stranded or double stranded form.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(for example, rRNA, tRNA and mRNA) or a defined sequence of amino acidsand the biological properties resulting therefrom. Thus, a gene encodesa protein if transcription and translation of mRNA produced by that geneproduces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and non-codingstrand, used as the template for transcription, of a gene or cDNA can bereferred to as encoding the protein or other product of that gene orcDNA. Unless otherwise specified, a “nucleotide sequence encoding anamino acid sequence” includes all nucleotide sequences that aredegenerate versions of each other and that encode the same amino acidsequence. Nucleotide sequences that encode proteins and RNA may includeintrons. In some examples, a nucleic acid encodes a disclosed PreFantigen.

Operably Linked:

A first nucleic acid sequence is operably linked with a second nucleicacid sequence when the first nucleic acid sequence is placed in afunctional relationship with the second nucleic acid sequence. Forinstance, a promoter is operably linked to a coding sequence if thepromoter affects the transcription or expression of the coding sequence.Generally, operably linked DNA sequences are contiguous and, wherenecessary to join two protein-coding regions, in the same reading frame.

Polypeptide:

Any chain of amino acids, regardless of length or post-translationalmodification (such as glycosylation or phosphorylation). “Polypeptide”applies to amino acid polymers including naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer as well as inwhich one or more amino acid residue is a non-natural amino acid, forexample an artificial chemical mimetic of a corresponding naturallyoccurring amino acid. A “residue” refers to an amino acid or amino acidmimetic incorporated in a polypeptide by an amide bond or amide bondmimetic. A polypeptide has an amino terminal (N-terminal) end and acarboxy terminal (C-terminal) end. “Polypeptide” is used interchangeablywith peptide or protein, and is used interchangeably herein to refer toa polymer of amino acid residues.

In many instances, a polypeptide folds into a specific three-dimensionalstructure, and can include surface-exposed amino acid residues andnon-surface-exposed amino acid residues. In some instances a protein caninclude multiple polypeptides that fold together into a functional unit.For example, the RSV F protein is composed of F₁/F₂ heterodimers thattrimerize in to a multimeric protein. “Surface-exposed amino acidresidues” are those amino acids that have some degree of exposure on thesurface of the protein, for example such that they can contact thesolvent when the protein is in solution. In contrast,non-surface-exposed amino acids are those amino acid residues that arenot exposed on the surface of the protein, such that they do not contactsolution when the protein is in solution. In some examples, thenon-surface-exposed amino acid residues are part of the protein core.

A “protein core” is the interior of a folded protein, which issubstantially free of solvent exposure, such as solvent in the form ofwater molecules in solution. Typically, the protein core ispredominately composed of hydrophobic or apolar amino acids. In someexamples, a protein core may contain charged amino acids, for exampleaspartic acid, glutamic acid, arginine, and/or lysine. The inclusion ofuncompensated charged amino acids (a compensated charged amino can be inthe form of a salt bridge) in the protein core can lead to adestabilized protein. That is, a protein with a lower T_(m) then asimilar protein without an uncompensated charged amino acid in theprotein core. In other examples, a protein core may have a cavity withinthe protein core. Cavities are essentially voids within a folded proteinwhere amino acids or amino acid side chains are not present. Suchcavities can also destabilize a protein relative to a similar proteinwithout a cavity. Thus, when creating a stabilized form of a protein, itmay be advantageous to substitute amino acid residues within the core inorder to fill cavities present in the wild-type protein.

Amino acids in a peptide, polypeptide or protein generally arechemically bound together via amide linkages (CONH). Additionally, aminoacids may be bound together by other chemical bonds. For example,linkages for amino acids or amino acid analogs can include CH₂NH—,—CH₂S—, —CH₂—CH₂, —CH═CH—(cis and trans), —COCH₂—, —CH(OH)CH₂—, and—CHH₂SO— (These and others can be found in Spatola, in Chemistry andBiochemistry of Amino Acids, Peptides, and Proteins, B. Weinstein, eds.,Marcel Dekker, New York, p. 267 (1983); Spatola, A. F., Vega Data (March1983), Vol. 1, Issue 3, Peptide Backbone Modifications (general review);Morley, Trends Pharm Sci pp. 463-468, 1980; Hudson, et al., Int J PeptProt Res 14:177-185, 1979; Spatola et al. Life Sci 38:1243-1249, 1986;Harm J. Chem. Soc Perkin Trans. 1307-314, 1982; Almquist et al. J. Med.Chem. 23:1392-1398, 1980; Jennings-White et al. Tetrahedron Lett23:2533, 1982; Holladay et al. Tetrahedron. Lett 24:4401-4404, 1983; andHruby Life Sci 31:189-199, 1982.

Peptide Modifications:

Peptides, such as the disclosed RSV F proteins stabilized in a prefusionconformation can be modified by a variety of chemical techniques toproduce derivatives having essentially the same activity andconformation as the unmodified peptides, and optionally having otherdesirable properties. For example, carboxylic acid groups of theprotein, whether carboxyl-terminal or side chain, may be provided in theform of a salt of a pharmaceutically-acceptable cation or esterified toform a C₁-C₁₆ ester, or converted to an amide of formula NR₁R₂ whereinR₁ and R₂ are each independently H or C₁-C₁₆ alkyl, or combined to forma heterocyclic ring, such as a 5- or 6-membered ring. Amino groups ofthe peptide, whether amino-terminal or side chain, may be in the form ofa pharmaceutically-acceptable acid addition salt, such as the HCl, HBr,acetic, benzoic, toluene sulfonic, maleic, tartaric and other organicsalts, or may be modified to C₁-C₁₆ alkyl or dialkyl amino or furtherconverted to an amide.

Hydroxyl groups of the peptide side chains can be converted to C₁-C₁₆alkoxy or to a C₁-C₁₆ ester using well-recognized techniques. Phenyl andphenolic rings of the peptide side chains can be substituted with one ormore halogen atoms, such as F, Cl, Br or I, or with C₁-C₁₆ alkyl, C₁-C₁₆alkoxy, carboxylic acids and esters thereof, or amides of suchcarboxylic acids. Methylene groups of the peptide side chains can beextended to homologous C₂-C₄ alkylenes. Thiols can be protected with anyone of a number of well-recognized protecting groups, such as acetamidegroups.

Pharmaceutically Acceptable Carriers:

The pharmaceutically acceptable carriers useful in this disclosure areconventional. Remington's Pharmaceutical Sciences, by E. W. Martin, MackPublishing Co., Easton, Pa., 19th Edition (1995), describes compositionsand formulations suitable for pharmaceutical delivery of the proteinsand other compositions herein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions, powder, pill, tablet, or capsule forms,conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

Prime-Boost Vaccination:

An immunotherapy including administration of a first immunogeniccomposition (the primer vaccine) followed by administration of a secondimmunogenic composition (the booster vaccine) to a subject to induce animmune response. The primer vaccine and the booster vaccine include avector (such as a viral vector or DNA vector) expressing the antigen towhich the immune response is directed. The booster vaccine isadministered to the subject after the primer vaccine; the skilledartisan will understand a suitable time interval between administrationof the primer vaccine and the booster vaccine, and examples of suchtimeframes are disclosed herein. In some embodiments, the primervaccine, the booster vaccine, or both primer vaccine and the boostervaccine additionally include an adjuvant.

Protein Nanoparticle:

A multi-subunit, protein-based polyhedron shaped structure. The subunitsare each composed of proteins or polypeptides (for example aglycosylated polypeptide), and, optionally of single or multiplefeatures of the following: nucleic acids, prosthetic groups, organic andinorganic compounds. Non-limiting examples of protein nanoparticlesinclude ferritin nanoparticles (see, e.g., Zhang, Y. Int. J. Mol. Sci.,12:5406-5421, 2011, incorporated by reference herein), encapsulinnanoparticles (see, e.g., Sutter et al., Nature Struct. and Mol. Biol.,15:939-947, 2008, incorporated by reference herein), Sulfur OxygenaseReductase (SOR) nanoparticles (see, e.g., Urich et al., Science,311:996-1000, 2006, incorporated by reference herein), lumazine synthasenanoparticles (see, e.g., Zhang et al., J. Mol. Biol., 306: 1099-1114,2001) or pyruvate dehydrogenase nanoparticles (see, e.g., Izard et al.,PNAS 96: 1240-1245, 1999, incorporated by reference herein). Ferritin,encapsulin, SOR, lumazine synthase, and pyruvate dehydrogenase aremonomeric proteins that self-assemble into a globular protein complexesthat in some cases consists of 24, 60, 24, 60, and 60 protein subunits,respectively. In some examples, ferritin, encapsulin, SOR, lumazinesynthase, or pyruvate dehydrogenase monomers are linked to a disclosedantigen (for example, a recombinant RSV F protein stabilized in aprefusion conformation) and self-assembled into a protein nanoparticlepresenting the disclosed antigens on its surface, which can beadministered to a subject to stimulate an immune response to theantigen.

Recombinant:

A recombinant nucleic acid is one that has a sequence that is notnaturally occurring or has a sequence that is made by an artificialcombination of two otherwise separated segments of sequence. Thisartificial combination can be accomplished by chemical synthesis or,more commonly, by the artificial manipulation of isolated segments ofnucleic acids, for example, by genetic engineering techniques. Arecombinant protein is a protein encoded by a heterologous (for example,recombinant) nucleic acid that has been introduced into a host cell,such as a bacterial or eukaryotic cell. The nucleic acid can beintroduced, for example, on an expression vector having signals capableof expressing the protein encoded by the introduced nucleic acid or thenucleic acid can be integrated into the host cell chromosome.

Repacking Amino Acid Substitution:

An amino acid substitution that increases the interactions ofneighboring residues in a protein, for example, by enhancing hydrophobicinteractions or hydrogen-bond formation, or by reducing unfavorable orrepulsive interactions of neighboring residues, for example, byeliminating clusters of similarly charged residues. In severalembodiments, a repacking amino acid substitution is introduced toincrease the interactions of neighboring residues in the RSV F proteinprefusion conformation, that are not in close proximity in the RSV Fpostfusion conformation. Typically, introduction of a repacking aminoacid substitution will increase the T_(m) of the prefusion conformationof the RSV F protein, and lower the T_(m) of the postfusion conformationof the RSV F protein.

Respiratory Syncytial Virus (RSV):

An enveloped non-segmented negative-sense single-stranded RNA virus ofthe family Paramyxoviridae. It is the most common cause of bronchiolitisand pneumonia among children in their first year of life and infectsnearly all children by 3 years of age. RSV also causes repeatedinfections including severe lower respiratory tract disease, which mayoccur at any age, especially among the elderly or those with compromisedcardiac, pulmonary, or immune systems. In the US, RSV bronchiolitis isthe leading cause of hospitalization in infants and a major cause ofasthma and wheezing throughout childhood (Shay et al., JAMA, 282, 1440(1999); Hall et al., N. Engl. J. Med., 360, 588 (2009)). Globally, RSVis responsible for 66,000-199,000 deaths each year for children youngerthan five years of age (Nair et al., Lancet, 375, 1545 (2010)), andaccounts for 6.7% of deaths among infants one month to one year old—morethan any other single pathogen except malaria (Lozano et al., Lancet,380, 2095 (2013)).

The RSV genome is ˜15,000 nucleotides in length and includes 10 genesencoding 11 proteins, including the glycoproteins SH, G and F. The Fprotein mediates fusion, allowing entry of the virus into the cellcytoplasm and also promoting the formation of syncytia. Two subtypes ofhuman RSV strains have been described, the A and B subtypes, based ondifferences in the antigenicity of the G glycoprotein. RSV strains forother species are also known, including bovine RSV. Exemplary RSV strainsequences are known to the person of ordinary skill in the art. Further,several models of human RSV infection are available, including modelorganisms infected with hRSV, as well as model organisms infected withspecies specific RSV, such as use of bRSV infection in cattle (see,e.g., Bern et al., Am J, Physiol. Lung Cell Mol. Physiol., 301:L148-L156, 2011).

Several methods of diagnosing RSV infection are known, including use ofDirect Fluorescent Antibody detection (DFA), Chromatographic rapidantigen detection, and detection of viral RNA using RT PCR.Quantification of viral load can be determined, for example, by PlaqueAssay, antigen capture enzyme immunoassay (EIA), ELISA and HA, andquantification of antibody levels by HAI and Neutralization assay.Current RSV treatment is passive administration of the monoclonalantibody palivizumab (SYNAGIS®), which recognizes the RSV F protein(Johnson et al., J. Infect. Dis., 176, 1215 (1997); Beeler and van WykeCoelingh, J. Virol., 63, 2941 (1989)) and reduces incidence of severedisease (The IMpact-RSV Study Group, Pediatrics, 102, 531 (1998)). (Alsosee, e.g., Nam and Kun (Eds.). Respiratory Syncytial Virus: Prevention,Diagnosis and Treatment. Nova Biomedical Nova Science Publisher, 2011;and Cane (Ed.) Respiratory Syncytial Virus. Elsevier Science, 2007.)

RSV Fusion (F) Protein:

An RSV envelope glycoprotein that facilitates fusion of viral andcellular membranes. In nature, the RSV F protein is initiallysynthesized as a single polypeptide precursor approximately 574 aminoacids in length, designated F₀. F₀ includes an N-terminal signal peptidethat directs localization to the endoplasmic reticulum, where the signalpeptide (approximately the first 25 residues of F₀) is proteolyticallycleaved. The remaining F₀ residues oligomerize to form a trimer which isagain proteolytically processed by a cellular protease at two conservedfurin consensus cleavage sequences (approximately F₀ positions 109 and136; for example, RARR₁₀₉ (SEQ ID NO: 124, residues 106-109) and RKRR₁₃₆(SEQ ID NO: 124, residues 133-136) releasing a 27 amino acidglycopeptide and generating two disulfide-linked fragments, F₁ and F₂.The smaller of these fragments, F₂, originates from the N-terminalportion of the F₀ precursor and includes approximately residues 26-109of F₀. The larger of these fragments, F₁, includes the C-terminalportion of the F₀ precursor (approximately residues 137-574) includingan extracellular/lumenal region (˜residues 137-524), a transmembranedomain (˜residues 525-550), and a cytoplasmic domain (˜residues 551-574)at the C-terminus.

Three F₂-F₁ protomers oligomerize to form a mature F protein, whichadopts a metastable “prefusion” conformation that is triggered toundergo a conformational change (to a “postfusion” conformation) uponcontact with a target cell membrane. This conformational change exposesa hydrophobic sequence, known as the fusion peptide, which is located atthe N-terminus of the F₁ polypeptide, and which associates with the hostcell membrane and promotes fusion of the membrane of the virus, or aninfected cell, with the target cell membrane.

A number of neutralizing antibodies that specifically bind to antigenicsites on RSV F protein have been identified. These include monoclonalantibodies 131-2a and 2F, which bind to antigenic site I (centeredaround residue P389); monoclonal antibodies palivizumab and motavizumab,which bind to antigenic site II (centered around residues 254-277); andmonoclonal antibodies 101F and mAb19, which bind to antigenic site IV(centered around residues 429-437).

RSV F₀ Polypeptide (F₀):

The precursor of the RSV F protein, including the amino acids of aN-terminal signal peptide, a F₂ polypeptide, a pep27 polypeptide, and aF₁ polypeptide including the F₁ extracellular domain, transmembranedomain and cytosolic tail. The native F₀ polypeptide is proteolyticallyprocessed at a signal sequence cleavage site, and two furin cleavagesites (approximately F₀ positions 109 and 136; for example, RARR₁₀₉ (SEQID NO: 124, residues 106-109) and RKRR₁₃₆ (SEQ ID NO: 124, residues133-136), resulting in the F₁ and F₂ fragments. Examples of F₀polypeptides from many different RSV subgroups are known, including fromthe A, B and bovine subgroups, examples of which are set forth herein asSEQ ID NOs: 1-128, 129-177, and 178-184, respectively.

RSV F₁ polypeptide (F₁): A peptide chain of the RSV F protein. As usedherein, “F₁ polypeptide” refers to both native F₁ polypeptides and F₁polypeptides including modifications (e.g., amino acid substitutions)from the native sequence, for example, modifications designed tostabilize a recombinant F protein (including the modified F₁polypeptide) in a RSV F protein prefusion conformation. Native F₁includes approximately residues 137-574 of the RSV F₀ precursor, andincludes (from N- to C-terminus) an extracellular/lumenal region(˜residues 137-524), a transmembrane domain (˜residues 525-550), and acytoplasmic domain (˜residues 551-574). Several embodiments include anF₁ polypeptide modified from a native F₁ sequence, for example an F₁polypeptide that lacks the transmembrane and cytosolic domain, orincludes one or more amino acid substitutions that stabilize arecombinant F protein (containing the F₁ polypeptide) in a prefusionconformation. In one example, a disclosed RSV F protein includes a F₁polypeptide with deletion of the transmembrane and cytosolic domains,cysteine substitutions at positions 155 and 290, and which includes aC-terminal linkage to a trimerization domain. Many examples of native F₁sequences are known which are provided herein as approximately positions137-524 of SEQ ID NOs: 1-184.

RSV F₂ Polypeptide (F₂):

A polypeptide chain of the RSV F protein. As used herein, “F₂polypeptide” refers to both native F₂ polypeptides and F₂ polypeptidesincluding modifications (e.g., amino acid substitutions) from the nativesequence, for example, modifications designed to stabilize a recombinantF protein (including the modified F₂ polypeptide) in a RSV F proteinprefusion conformation. Native F₂ includes approximately residues 26-109of the RSV F₀ precursor. In native RSV F protein, the F₂ polypeptide islinked to the F₁ polypeptide by two disulfide bonds. Many examples ofnative F₂ sequences are known which are provided herein as approximatelypositions 26-109 of SEQ ID NOs: 1-184.

RSV Pep27 Polypeptide (Pep27):

A 27 amino acid polypeptide that is excised from the F₀ precursor duringmaturation of the RSV F protein. pep27 is flanked by two furin cleavagesites that are cleaved by a cellular protease during F proteinmaturation to generate the F₁ and F₂ polypeptide. Examples of nativepep27 sequences are known which are provided herein as positions 110-136of SEQ ID NOs: 1-184.

RSV F Protein Prefusion Conformation:

A structural conformation adopted by the RSV F protein prior totriggering of the fusogenic event that leads to transition of RSV F tothe postfusion conformation. The three-dimensional structure of anexemplary RSV F protein in a prefusion conformation is disclosed herein(see Example 1) and the structural coordinates of the exemplary RSV Fprotein in a prefusion conformation bound by the prefusion specificantibody D25 are provided in Table 1. In the prefusion state, the RSV Fprotein includes an antigenic site at the membrane distal apex(“antigenic site Ø,” see Example 1), that includes the epitopes of theD25 and AM22 antibodies. As used herein, a recombinant RSV F proteinstabilized in a prefusion conformation can be specifically bound by anantibody that is specific for the prefusion conformation of the RSV Fprotein, such as an antibody that specifically binds to an epitopewithin antigenic site Ø, for example, the D25 or AM22 antibody.

RSV F Protein Postfusion Conformation:

A structural conformation adopted by the RSV F protein that is not theprefusion conformation. The post fusion conformation of RSV F proteinhas been described at the atomic level (see, e.g., McLellan et al., J.Virol., 85, 7788, 2011; Swanson et al., Proc. Natl. Acad. Sci. U.S.A.,108, 9619, 2011; and structural coordinates deposited PDB Accession No.3RRR; each of which is incorporated by reference herein). In thepostfusion conformation, the RSV F protein does not include antigenicsite Ø, and therefore does not include the D25 epitope and is notspecifically bound by D25 or AM22. The RSV postfusion conformationoccurs, for example, following fusion of the F protein with the cellmembrane.

Resurfaced Antigen or Resurfaced Immunogen:

A polypeptide immunogen derived from a wild-type antigen in which aminoacid residues outside or exterior to a target epitope are mutated in asystematic way to focus the immunogenicity of the antigen to theselected target epitope. In some examples a resurfaced antigen isreferred to as an antigenically-cloaked immunogen orantigenically-cloaked antigen.

Root Mean Square Deviation (RMSD):

The square root of the arithmetic mean of the squares of the deviationsfrom the mean. In several embodiments, RMSD is used as a way ofexpressing deviation or variation from the structural coordinates of areference three dimensional structure. This number is typicallycalculated after optimal superposition of two structures, as the squareroot of the mean square distances between equivalent C_(α) atoms. Insome embodiments, the reference three-dimensional structure includes thestructural coordinates of the RSV F protein bound to monoclonal antibodyD25, set forth herein in Table 1.

Sequence Identity/Similarity:

The identity/similarity between two or more nucleic acid sequences, ortwo or more amino acid sequences, is expressed in terms of the identityor similarity between the sequences. Sequence identity can be measuredin terms of percentage identity; the higher the percentage, the moreidentical the sequences are. Homologs or orthologs of nucleic acid oramino acid sequences possess a relatively high degree of sequenceidentity/similarity when aligned using standard methods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smith &Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol.Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp,CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988;Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; andPearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J.Mol. Biol. 215:403-10, 1990, presents a detailed consideration ofsequence alignment methods and homology calculations.

Once aligned, the number of matches is determined by counting the numberof positions where an identical nucleotide or amino acid residue ispresent in both sequences. The percent sequence identity is determinedby dividing the number of matches either by the length of the sequenceset forth in the identified sequence, or by an articulated length (suchas 100 consecutive nucleotides or amino acid residues from a sequenceset forth in an identified sequence), followed by multiplying theresulting value by 100. For example, a peptide sequence that has 1166matches when aligned with a test sequence having 1554 amino acids is75.0 percent identical to the test sequence (1166÷1554*100=75.0). Thepercent sequence identity value is rounded to the nearest tenth. Forexample, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The lengthvalue will always be an integer.

For sequence comparison of nucleic acid sequences and amino acidssequences, typically one sequence acts as a reference sequence, to whichtest sequences are compared. When using a sequence comparison algorithm,test and reference sequences are entered into a computer, subsequencecoordinates are designated, if necessary, and sequence algorithm programparameters are designated. Default program parameters are used. Methodsof alignment of sequences for comparison are well known in the art.Optimal alignment of sequences for comparison can be conducted, forexample, by the local homology algorithm of Smith & Waterman, Adv. Appl.Math. 2:482, 1981, by the homology alignment algorithm of Needleman &Wunsch, J. Mol. Biol. 48:443, 1970, by the search for similarity methodof Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by manual alignment andvisual inspection (see for example, Current Protocols in MolecularBiology (Ausubel et al., eds 1995 supplement)). The NCBI Basic LocalAlignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol.215:403-10, 1990) is available from several sources, including theNational Center for Biological Information (NCBI, National Library ofMedicine, Building 38A, Room 8N805, Bethesda, Md. 20894) and on theInternet, for use in connection with the sequence analysis programsblastp, blastn, blastx, tblastn, and tblastx. Blastn is used to comparenucleic acid sequences, while blastp is used to compare amino acidsequences. Additional information can be found at the NCBI web site.

Another example of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and the BLAST2.0 algorithm, which are described in Altschul et al., J. Mol. Biol.215:403-410, 1990 and Altschul et al., Nucleic Acids Res. 25:3389-3402,1977. Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information (World WideWeb address ncbi.nlm.nih.gov). The BLASTN program (for nucleotidesequences) uses as defaults a word length (W) of 11, alignments (B) of50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.The BLASTP program (for amino acid sequences) uses as defaults a wordlength (W) of 3, and expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915,1989).

Another indication of sequence similarity between two nucleic acids isthe ability to hybridize. The more similar are the sequences of the twonucleic acids, the more stringent the conditions at which they willhybridize. The stringency of hybridization conditions aresequence-dependent and are different under different environmentalparameters. Thus, hybridization conditions resulting in particulardegrees of stringency will vary depending upon the nature of thehybridization method of choice and the composition and length of thehybridizing nucleic acid sequences. Generally, the temperature ofhybridization and the ionic strength (especially the Na⁺ and/or Mg⁺concentration) of the hybridization buffer will determine the stringencyof hybridization, though wash times also influence stringency.Generally, stringent conditions are selected to be about 5° C. to 20° C.lower than the thermal melting point (T_(m)) for the specific sequenceat a defined ionic strength and pH. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of the target sequencehybridizes to a perfectly matched probe. Conditions for nucleic acidhybridization and calculation of stringencies can be found, for example,in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Tijssen,Hybridization With Nucleic Acid Probes, Part I. Theory and Nucleic AcidPreparation, Laboratory Techniques in Biochemistry and MolecularBiology, Elsevier Science Ltd., NY, N.Y., 1993; and Ausubel et al. ShortProtocols in Molecular Biology, 4^(th) ed., John Wiley & Sons, Inc.,1999.

“Stringent conditions” encompass conditions under which hybridizationwill only occur if there is less than 25% mismatch between thehybridization molecule and the target sequence. “Stringent conditions”may be broken down into particular levels of stringency for more precisedefinition. Thus, as used herein, “moderate stringency” conditions arethose under which molecules with more than 25% sequence mismatch willnot hybridize; conditions of “medium stringency” are those under whichmolecules with more than 15% mismatch will not hybridize, and conditionsof “high stringency” are those under which sequences with more than 10%mismatch will not hybridize. Conditions of “very high stringency” arethose under which sequences with more than 6% mismatch will nothybridize. In contrast nucleic acids that hybridize under “lowstringency conditions include those with much less sequence identity, orwith sequence identity over only short subsequences of the nucleic acid.

Signal Peptide:

A short amino acid sequence (e.g., approximately 18-25 amino acids inlength) that directs newly synthesized secretory or membrane proteins toand through membranes (for example, the endoplasmic reticulum membrane).Signal peptides are typically located at the N-terminus of a polypeptideand are removed by signal peptidases after the polypeptide has crossedthe membrane. Signal peptide sequences typically contain three commonstructural features: an N-terminal polar basic region (n-region), ahydrophobic core, and a hydrophilic c-region). Exemplary signal peptidesequences are set forth as residues 1-25 of SEQ ID NOs: 1-182 (RSV Fprotein signal peptides from A, B, and bovine RSV).

Specifically Bind:

When referring to the formation of an antibody:antigen protein complex,refers to a binding reaction which determines the presence of a targetprotein, peptide, or polysaccharide (for example a glycoprotein), in thepresence of a heterogeneous population of proteins and other biologics.Thus, under designated conditions, an antibody binds preferentially to aparticular target protein, peptide or polysaccharide (such as an antigenpresent on the surface of a pathogen, for example RSV F) and does notbind in a significant amount to other proteins or polysaccharidespresent in the sample or subject. An antibody that specifically binds tothe prefusion conformation of RSV F protein (e.g., and antibody thatspecifically binds to antigenic site Ø) does not specifically bind tothe postfusion conformation of RSV F protein. Specific binding can bedetermined by methods known in the art. With reference to anantibody:antigen or Fab:antigen complex, specific binding of the antigenand antibody has a K_(d)(or apparent K_(d)) of less than about 10⁻⁶,such as less than about 10⁻⁷ Molar, 10⁻⁸ Molar, 10⁻⁹, or even less thanabout 10⁻¹⁰ Molar.

Soluble Protein:

A protein capable of dissolving in aqueous liquid at room temperatureand remaining dissolved. The solubility of a protein may changedepending on the concentration of the protein in the water-based liquid,the buffering condition of the liquid, the concentration of othersolutes in the liquid, for example salt and protein concentrations, andthe heat of the liquid. In several embodiments, a soluble protein is onethat dissolves to a concentration of at least 0.5 mg/ml in phosphatebuffered saline (pH 7.4) at room temperature and remains dissolved forat least 48 hours.

Therapeutic Agent:

A chemical compound, small molecule, or other composition, such asnucleic acid molecule, capable of inducing a desired therapeutic orprophylactic effect when properly administered to a subject.

Therapeutically Effective Amount or Effective Amount:

The amount of agent, such as a disclosed antigen or immunogeniccomposition containing a disclosed antigen, that is sufficient toprevent, treat (including prophylaxis), reduce and/or ameliorate thesymptoms and/or underlying causes of any of a disorder or disease, forexample to prevent, inhibit, and/or treat RSV infection. In someembodiments, an “effective amount” is sufficient to reduce or eliminatea symptom of a disease, such as RSV infection. For instance, this can bethe amount necessary to inhibit viral replication or to measurably alteroutward symptoms of the viral infection. In general, this amount will besufficient to measurably inhibit virus (for example, RSV) replication orinfectivity. When administered to a subject, a dosage will generally beused that will achieve target tissue concentrations that has been shownto achieve in vitro inhibition of viral replication. It is understoodthat to obtain a protective immune response against a pathogen canrequire multiple administrations of the immunogenic composition. Thus, atherapeutically effective amount encompasses a fractional dose thatcontributes in combination with previous or subsequent administrationsto attaining a protective immune response.

Transmembrane Domain:

An amino acid sequence that inserts into a lipid bilayer, such as thelipid bilayer of a cell or virus or virus-like particle. A transmembranedomain can be used to anchor an antigen to a membrane. In some examplesa transmembrane domain is a RSV F protein transmembrane domain.Exemplary RSV F transmembrane domains are familiar to the person ofordinary skill in the art, and provided herein. For example, the aminoacid sequences of exemplary RSV F transmembrane domains are provided aspositions 525-550 of SEQ ID NOs: 1-183.

Transformed:

A transformed cell is a cell into which has been introduced a nucleicacid molecule by molecular biology techniques. As used herein, the termtransformation encompasses all techniques by which a nucleic acidmolecule might be introduced into such a cell, including transfectionwith viral vectors, transformation with plasmid vectors, andintroduction of DNA by electroporation, lipofection, and particle gunacceleration.

Vaccine:

A pharmaceutical composition that elicits a prophylactic or therapeuticimmune response in a subject. In some cases, the immune response is aprotective immune response. Typically, a vaccine elicits anantigen-specific immune response to an antigen of a pathogen, forexample a viral pathogen, or to a cellular constituent correlated with apathological condition. A vaccine may include a polynucleotide (such asa nucleic acid encoding a disclosed antigen), a peptide or polypeptide(such as a disclosed antigen), a virus, a cell or one or more cellularconstituents.

Vector:

A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. Recombinant DNA vectors are vectorshaving recombinant DNA. A vector can include nucleic acid sequences thatpermit it to replicate in a host cell, such as an origin of replication.A vector can also include one or more selectable marker genes and othergenetic elements known in the art. Viral vectors are recombinant DNAvectors having at least some nucleic acid sequences derived from one ormore viruses.

A replication deficient viral vector that requires complementation ofone or more regions of the viral genome required for replication, as aresult of, for example a deficiency in at least onereplication-essential gene function. For example, such that the viralvector does not replicate in typical host cells, especially those in ahuman patient that could be infected by the viral vector in the courseof a therapeutic method. Examples of replication-deficient viral vectorsand systems for their use are known in the art and include; for examplereplication-deficient LCMV vectors (see, e.g., U.S. Pat. Pub. No.2010/0297172, incorporated by reference herein in its entirety) andreplication deficient adenoviral vectors (see, e.g., PCT App. Pub. No.WO2000/00628, incorporated by reference herein).

Virus:

A virus consists essentially of a core of nucleic acid surrounded by aprotein coat, and has the ability to replicate only inside a livingcell. “Viral replication” is the production of additional virus by theoccurrence of at least one viral life cycle. A virus may subvert thehost cells' normal functions, causing the cell to behave in a mannerdetermined by the virus. For example, a viral infection may result in acell producing a cytokine, or responding to a cytokine, when theuninfected cell does not normally do so. In some examples, a virus is apathogen.

Virus-Like Particle (VLP):

A non-replicating, viral shell, derived from any of several viruses.VLPs are generally composed of one or more viral proteins, such as, butnot limited to, those proteins referred to as capsid, coat, shell,surface and/or envelope proteins, or particle-forming polypeptidesderived from these proteins. VLPs can form spontaneously uponrecombinant expression of the protein in an appropriate expressionsystem. Methods for producing particular VLPs are known in the art. Thepresence of VLPs following recombinant expression of viral proteins canbe detected using conventional techniques known in the art, such as byelectron microscopy, biophysical characterization, and the like.Further, VLPs can be isolated by known techniques, e.g., densitygradient centrifugation and identified by characteristic densitybanding. See, for example, Baker et al. (1991) Biophys. J. 60:1445-1456;and Hagensee et al. (1994) J. Virol. 68:4503-4505; Vincente, J InvertebrPathol., 2011; Schneider-Ohrum and Ross, Curr. Top. Microbiol. Immunol.,354: 53073, 2012).

II. DESCRIPTION OF SEVERAL EMBODIMENTS

It is disclosed herein that the RSV F protein undergoes a dramaticstructural rearrangement between its pre- and postfusion conformations(see Example 1, below). As shown in FIG. 2B, the N-terminal region ofthe F₁ polypeptide in the prefusion conformation (corresponding in partto the membrane distal lobe shown in FIG. 2A) includes the indicated α2,α3, β3, β4, and α4 helical and beta sheet structures, whereas thecorresponding region of the N-terminus of the F₁ polypeptide in thepostfusion structure includes an extended α5 helical structure. Further,the C-terminal region of the F₁ polypeptide in the prefusionconformation (corresponding in part to the membrane proximal lobe shownin FIG. 2A) includes the indicated (β22, α9, and β23 beta sheet andhelical structures, whereas the corresponding C-terminal region of theof the F₁ polypeptide in the postfusion conformation structure includesan extended α10 helical structure. Thus, the membrane distal andmembrane proximal lobes of the RSV F protein in its prefusionconformation include several distinct structural elements that areabsent from the corresponding regions of the RSV F protein in itspostfusion conformation. Amino acid positions (and sequences)corresponding to these regions are highlighted in grey in FIG. 2,including positions 137-216, and 461-513 of the F₁ polypeptide.

RSV F protein antigens are provided that are stabilized or “locked” in aprefusion conformation, termed “PreF antigens.” Using structure-guideddesign, positions of the RSV F₁ and F₂ polypeptides are targeted formodification (e.g., amino acid substitution) to hinder or preventtransition of the RSV F protein from a pre- to postfusion conformation.Such antigens have utility, for example, as immunogens to induce aneutralizing antibody response to RSV F protein.

A. Native RSV F Proteins

Native RSV F proteins from different RSV groups, as well as nucleic acidsequences encoding such proteins and methods, are known. For example,the sequence of several group A (Seq_(—)1-128), B (Seq_(—)129-177) andbovine (Seq_(—)178-184) precursor RSV F₀ proteins provided as SEQ IDNOs: 1-184. The GenInfo Identifier (gi) and corresponding accessionnumber for each of these sequences, as well as the corresponding RSVgroup are provided in Table 3, below:

TABLE 3 Exemplary Group A, B and bovine RSV F protein sequences SEQAccession 1 gi|113472470|gb|ABI35685.1 2 gi|46405966|gb|AAS93651.1 3gi|346682949|gb|AEO45830.1 4 gi|392301680|gb|AFM55244.1 5gi|392301896|gb|AFM55442.1 6 gi|392301692|gb|AFM55255.1 7gi|392301728|gb|AFM55288.1 8 gi|392976459|gb|AFM95385.1 9gi|392976475|gb|AFM95400.1 10 gi|21689583|gb|AAM68157.1 11gi|21689587|gb|AAM68160.1 12 gi|346682981|gb|AEO45859.1 13gi|352962949|gb|AEQ63444.1 14 gi|353441614|gb|AEQ98752.1 15gi|392301740|gb|AFM55299.1 16 gi|346682971|gb|AEO45850.1 17gi|346682992|gb|AEO45869.1 18 gi|346683003|gb|AEO45879.1 19gi|346683036|gb|AEO45909.1 20 gi|21689579|gb|AAM68154.1 21gi|326578296|gb|ADZ95777.1 22 gi|330470871|gb|AEC32087.1 23gi|346683058|gb|AEO45929.1 24 gi|392301644|gb|AFM55211.1 25gi|392301656|gb|AFM55222.1 26 gi|392301776|gb|AFM55332.1 27gi|46405962|gb|AAS93649.1 28 gi|326578298|gb|ADZ95778.1 29gi|392301872|gb|AFM55420.1 30 gi|346682960|gb|AEO45840.1 31gi|346683080|gb|AEO45949.1 32 gi|227299|prf||1701388A/1-574 33gi|352962996|gb|AEQ63487.1 34 gi|352963032|gb|AEQ63520.1 35gi|46405970|gb|AAS93653.1 36 gi|392976437|gb|AFM95365.1 37gi|392976449|gb|AFM95376.1 38 gi|352962805|gb|AEQ63312.1 39gi|346340362|gb|AEO23051.1 40 gi|352962829|gb|AEQ63334.1 41gi|352962865|gb|AEQ63367.1 42 gi|392302028|gb|AFM55563.1 43gi|392302016|gb|AFM55552.1 44 gi|417346971|gb|AFX60137.1 45gi|417347051|gb|AFX60173.1 46 gi|392301812|gb|AFM55365.1 47gi|29290039|gb|AAO72323.1 48 gi|29290041|gb|AAO72324.1 49gi|262479010|gb|ACY68435.1 50 gi|330470867|gb|AEC32085.1 51gi|392301704|gb|AFM55266.1 52 gi|392301716|gb|AFM55277.1 53gi|392301800|gb|AFM55354.1 54 gi|345548062|gb|AEO12131.1 55gi|346340367|gb|AEO23052.1 56 gi|352962889|gb|AEQ63389.1 57gi|353441606|gb|AEQ98748.1 58 gi|353441604|gb|AEQ98747.1 59gi|353441608|gb|AEQ98749.1 60 gi|353441616|gb|AEQ98753.1 61gi|353441620|gb|AEQ98755.1 62 gi|353441624|gb|AEQ98757.1 63gi|409905594|gb|AFV46409.1 64 gi|409905610|gb|AFV46417.1 65gi|417346953|gb|AFX60128.1 66 gi|417347079|gb|AFX60187.1 67gi|417346955|gb|AFX60129.1 68 gi|417346967|gb|AFX60135.1 69gi|417346979|gb|AFX60141.1 70 gi|417346993|gb|AFX60148.1 71gi|417346999|gb|AFX60151.1 72 gi|417347043|gb|AFX60169.1 73gi|417347105|gb|AFX60200.1 74 gi|417347107|gb|AFX60201.1 75gi|392301788|gb|AFM55343.1 76 gi|409905578|gb|AFV46401.1 77gi|409905596|gb|AFV46410.1 78 gi|353441622|gb|AEQ98756.1 79gi|409905582|gb|AFV46403.1 80 gi|417347109|gb|AFX60202.1 81gi|409905602|gb|AFV46413.1 82 gi|409905604|gb|AFV46414.1 83gi|417347121|gb|AFX60208.1 84 gi|409905614|gb|AFV46419.1 85gi|409905616|gb|AFV46420.1 86 gi|417346973|gb|AFX60138.1 87gi|417346997|gb|AFX60150.1 88 gi|417347021|gb|AFX60162.1 89gi|417347085|gb|AFX60190.1 90 gi|425706126|gb|AFX95851.1 91gi|392301836|gb|AFM55387.1 92 gi|392301992|gb|AFM55530.1 93gi|346683047|gb|AEO45919.1 94 gi|46405974|gb|AAS93655.1 95gi|46405976|gb|AAS93656.1 96 gi|346683069|gb|AEO45939.1 97gi|1353201|sp|P11209.2 98 gi|1912295|gb|AAC57027.1 99gi|9629375|ref|NP_044596.1 100 gi|21263086|gb|AAM44851.1 101gi|417346951|gb|AFX60127.1 102 gi|417347009|gb|AFX60156.1 103gi|29290043|gb|AAO72325.1 104 gi|138252|sp|P12568.1 105gi|226438|prf||1512372A 106 gi|37674744|gb|AAQ97026.1 107gi|37674754|gb|AAQ97031.1 108 gi|37674746|gb|AAQ97027.1 109gi|37674748|gb|AAQ97028.1 110 gi|37674750|gb|AAQ97029.1 111gi|37674752|gb|AAQ97030.1 112 gi|146738079|gb|ABQ42594.1 113gi|403379|emb|CAA81295.1 114 gi|226838116|gb|ACO83302.1 115gi|326578304|gb|ADZ95781.1 116 gi|326578306|gb|ADZ95782.1 117gi|326578308|gb|ADZ95783.1 118 gi|326578310|gb|ADZ95784.1 119gi|326578312|gb|ADZ95785.1 120 gi|60549171|gb|AAX23994.1 121gi|226838109|gb|ACO83297.1 122 gi|352962877|gb|AEQ63378.1 123gi|346683014|gb|AEO45889.1 124 gi|138251|sp|P03420.1| 125gi|1695263|gb|AAC55970.1 126 gi|61211|emb|CAA26143.1 127gi|226838114|gb|ACO83301.1 128 gi|352963080|gb|AEQ63564.1 129gi|109689536|dbj|BAE96918.1 130 gi|380235900|gb|AFD34266.1 131gi|401712638|gb|AFP99059.1 132 gi|401712648|gb|AFP99064.1 133gi|380235886|gb|AFD34259.1 134 gi|326578302|gb|ADZ95780.1 135gi|326578294|gb|ADZ95776.1 136 gi|326578300|gb|ADZ95779.1 137gi|380235892|gb|AFD34262.1 138 gi|46405984|gb|AAS93660.1 139gi|46405986|gb|AAS93661.1 140 gi|46405990|gb|AAS93663.1 141gi|46405992|gb|AAS93664.1 142 gi|345121421|gb|AEN74946.1 143gi|417347137|gb|AFX60215.1 144 gi|380235888|gb|AFD34260.1 145gi|346340378|gb|AEO23054.1 146 gi|384872848|gb|AFI25262.1 147gi|380235890|gb|AFD34261.1 148 gi|46405978|gb|AAS93657.1 149gi|46405982|gb|AAS93659.1 150 gi|352963104|gb|AEQ63586.1 151gi|352963128|gb|AEQ63608.1 152 gi|352963164|gb|AEQ63641.1 153gi|46405996|gb|AAS93666.1 154 gi|417347131|gb|AFX60212.1 155gi|417347135|gb|AFX60214.1 156 gi|417347145|gb|AFX60219.1 157gi|380235898|gb|AFD34265.1 158 gi|352963116|gb|AEQ63597.1 159gi|401712640|gb|AFP99060.1 160 gi|352963152|gb|AEQ63630.1 161gi|401712642|gb|AFP99061.1 162 gi|417347133|gb|AFX60213.1 163gi|417347147|gb|AFX60220.1 164 gi|417347151|gb|AFX60222.1 165gi|417347169|gb|AFX60231.1 166 gi|417347171|gb|AFX60232.1 167gi|417347175|gb|AFX60234.1 168 gi|46405988|gb|AAS93662.1 169gi|138250|sp|P13843.1 170 gi|2582041|gb|AAB82446.1 171gi|9629206|ref|NP_056863.1 172 gi|38230490|gb|AAR14266.1 173gi|326578292|gb|ADZ95775.1 174 gi|345121416|gb|AEN74944.1 175gi|345121418|gb|AEN74945.1 176 gi|46405994|gb|AAS93665.1 177gi|380235896|gb|AFD34264.1 178 gi|138247|sp|P22167.1 179gi|3451386|emb|CAA76980.1 180 gi|17939990|gb|AAL49399.1 181gi|9631275|ref|NP_048055.1 182 gi|94384139|emb|CA196787.1 183gi|425678|gb|AAB28458.1 184 gi|17940002|gb|AAL49410.1

The RSV F protein exhibits remarkable sequence conservation across RSVsubtypes (see Table 3, below, which shows average pairwise sequenceidentity across subtypes and F protein segments). For example, RSVsubtypes A and B share 90% sequence identity, and RSV subtypes A and Beach share 81% sequence identify with bRSV F protein, across the F₀precursor molecule. Within RSV subtypes the F₀ sequence identity is evengreater; for example within each of RSV A, B, and bovine subtypes, theRSV F₀ precursor protein has ˜98% sequence identity. Nearly allidentified RSV F₀ precursor proteins are approximately 574 amino acidsin length, with minor differences in length typically due to the lengthof the C-terminal cytoplasmic tail. Sequence identity across RSV Fproteins is illustrated in Table 4, below:

TABLE 4 RSV F protein sequence identity Sub-type hRSV A hRSV B bRSV hRSVA hRSV B bRSV hRSV A hRSV B bRSV F₀ (positions 1-574) F₁ (positions137-513) F₂ (positions 26-109) hRSV A 98% — — 99% — — 98% — — hRSV B 90%99% — 95% >99% — 93% 99% — bRSV 81% 81% 98% 91% 92% 99% 77% 77% 98%

In view of the conservation of RSV F sequences, the person of ordinaryskill in the art can easily compare amino acid positions betweendifferent native RSV F sequences, to identify corresponding RSV F aminoacid positions between different RSV strains and subtypes. For example,across nearly all identified native RSV F₀ precursor proteins, the furincleavage sites fall in the same amino acid positions. Thus, theconservation of RSV F protein sequences across strains and subtypesallows use of a reference RSV F sequence for comparison of amino acidsat particular positions in the RSV F protein. For the purposes of thisdisclosure (unless context indicates otherwise), RSV F protein aminoacid positions are given with reference to the reference F₀ proteinprecursor polypeptide set forth as SEQ ID NO: 124 (corresponding toGENBANK® Acc. No. P03420, incorporated by reference herein as present inGENBANK® on Feb. 28, 2013).

B. PreF Antigens

Isolated antigens are disclosed herein that include a recombinant RSV Fprotein stabilized in a prefusion conformation (“PreF antigens”). ThePreF antigens contain a recombinant RSV F protein that has been modifiedfrom a native form to increase immunogenicity. For example, thedisclosed recombinant RSV F proteins have been modified from the nativeRSV sequence to be stabilized in a prefusion conformation. The person ofordinary skill in the art will appreciate that the disclosed PreFantigens are useful to induce immunogenic responses in vertebrateanimals (such as mammals, for example, humans and cattle) to RSV (forexample RSV A, RSV B, or bovine RSV). Thus, in several embodiments, thedisclosed antigens are immunogens.

The D25 antibody recognizes a quaternary epitope including multipleprotomers of the RSV F protein. This epitope is contained within aantigenic site (“Antigenic site Ø”) located on the membrane-distal apexof the RSV F glycoprotein (see, e.g., FIG. 1C), when it is in aprefusion conformation. While the secondary structural elements of thethis epitope remains mostly unchanged between pre- and post-fusion Fconformations, their relative orientation changes substantially, withthe α4-helix pivoting ˜180° relative to strand β2 in pre- andpost-fusion conformations (see, e.g., FIG. 3B). The conformationalchanges in the structure of the RSV F protein between the pre- andpost-fusion conformations determines the presence of the D25 epitope onthe RSV F protein. Accordingly, in several embodiments, a PreF antigenincluding a recombinant RSV F protein stabilized in a prefusionconformation can be identified by determining the specific binding ofthe D25 monoclonal antibody to the antigen. The person of ordinary skillin the art will appreciate that other antibodies that specifically bindto antigenic site Ø of the RSV F protein (such as the AM22 antibody),can also be used to identify a PreF antigen including a RSV F proteinstabilized in a prefusion conformation.

Thus, the PreF antigens disclosed herein are specifically bound by anantibody that is specific for the RSV F prefusion conformation but notthe post fusion conformation. In several embodiments, the PreF antigenis specifically bound by the D25 and/or AM22 antibody, which (asdisclosed herein) are antibodies specific for the pre- but notpost-fusion conformation of the RSV F protein. In several examples, theprefusion specific antibody (such as D25 or AM22) specifically binds tothe PreF antigen with a dissociation constant of less than about 10⁻⁶Molar, such as less than about 10⁻⁷ Molar, 10⁻⁸ Molar, or less than 10⁻⁹Molar. Specific binding can be determined by methods known in the art.The determination of specific binding may readily be made by using oradapting routine procedures, such as ELISA, immunocompetition, surfaceplasmon resonance, or other immunosorbant assays (described in manystandard texts, including Harlow and Lane, Using Antibodies: ALaboratory Manual, CSHL, New York, 1999).

In further embodiments, the PreF antigen is not specifically bound by anantibody specific for the postfusion conformation of the RSV F protein.For example, an antibody specific for the six helix bundle found only inthe postfusion conformation of RSV F protein (e.g., as described inMagro et al., Proc. Nat'l. Acad. Sci. U.S.A., 109:3089-3094, 2012). Inseveral examples, the dissociation constant for the RSV F postfusionspecific antibody binding to the PreF antigen is greater than 10⁻⁵Molar, such as at least 10⁻⁵ Molar, 10⁻⁴ Molar, or 10⁻³.

In several embodiments, any of the PreF antigens includes a RSV Fprotein prefusion epitope (such as a D25 or AM22 epitope) in a RSV Fprotein prefusion specific antibody-bound conformation (such as a D25 orAM22 bound conformation). For example, in several embodiments, any ofthe PreF antigens includes an epitope in a D25 or AM22 epitope-boundconformation (e.g., the conformation defined by the structuralcoordinates provided in Table 1) when the PreF antigen is not bound byD25 or AM22, that is, the PreF antigen is stabilized in the D25- orAM22-bound conformation. Methods of determining if a disclosed PreFantigen includes a RSV F protein prefusion epitope (such as a D25 orAM22 epitope) in a RSV F protein prefusion specific monoclonalantibody-bound conformation (such as a D25 or AM22 bound conformation)are known to the person of ordinary skill in the art and furtherdisclosed herein (see, for example, McLellan et al., Nature,480:336-343, 2011; and U.S. Patent Application Publication No.2010/0068217, each of which is incorporated by reference herein in itsentirety). For example, the disclosed three-dimensional structure of theD25 Fab fragment in complex with the RSV F protein can be compared withthree-dimensional structure of any of the disclosed PreF antigens.

The person of ordinary skill in the art will appreciate that a disclosedPreF antigen can include an epitope in a prefusion specific monoclonalantibody-bound conformation even though the structural coordinates ofantigen are not strictly identical to those of the prefusion F proteinas disclosed herein. For example, in several embodiments, any of thedisclosed PreF antigens include a RSV F prefusion-specific epitope (suchas a D25 or AM22 epitope) that in the absence of the RSV F prefusionspecific monoclonal antibody can be structurally superimposed onto thecorresponding epitope in complex with the RSV F prefusion specificmonoclonal antibody with a root mean square deviation (RMSD) of theircoordinates of less than 1.0, 0.75, 0.5, 0.45, 0.4, 0.35, 0.3 or 0.25Å/residue, wherein the RMSD is measured over the polypeptide backboneatoms N, Cα, C, O, for at least three consecutive amino acids.

In several embodiments, the PreF antigen is soluble in aqueous solution.For example, in some embodiments, the PreF antigen is soluble in asolution that lacks detergent. in some embodiments, the PreF antigendissolves to a concentration of at least 0.5 mg/ml (such as at least 1.0mg/ml, 1.5 mg/ml, 2.0 mg/ml, 3.0 mg/ml, 4.0 mg/ml or at least 5.0 mg/ml)in phosphate buffered saline (pH 7.4) at room temperature (e.g., 20-22degrees Celsius) and remains dissolved for at least for at least 12hours (such as at least 24 hours, at least 48 hours, at least one week,at least two weeks, or more time). In one embodiment, the phosphatebuffered saline includes NaCl (137 mM), KCl (2.7 mM), Na₂HPO₄ (10 mM),KH₂PO₄ (1.8 mM) at pH 7.4. In some embodiments, the phosphate bufferedsaline further includes CaCl₂ (1 mM) and MgCl₂ (0.5 mM). The person ofskill in the art is familiar with methods of determining if a proteinremains in solution over time. For example, the concentration of theprotein dissolved in an aqueous solution can be tested over time usingstandard methods.

In several embodiments, any of the disclosed PreF antigens can be usedto induce an immune response to RSV in a subject. In several suchembodiments, induction of the immune response includes production ofneutralizing antibodies to RSV. Methods to assay for neutralizationactivity are known to the person of ordinary skill in the art andfurther described herein, and include, but are not limited to, plaquereduction neutralization (PRNT) assays, microneutralization assays (seee.g., Anderson et al., J. Clin. Microbiol., 22: 1050-1052, 1985), orflow cytometry based assays (see, e.g., Chen et al., J. Immunol.Methods., 362:180-184, 2010). Additional neutralization assays aredescribed herein, and familiar to the person of ordinary skill in theart.

In some embodiments, the PreF antigen includes a recombinant RSV Fprotein that, when dissolved in an aqueous solution, forms a populationof recombinant RSV F proteins stabilized in a prefusion conformation.The aqueous solution can be, for example, phosphate buffered saline atphysiological pH, such as pH 7.4. In some embodiments, the population isa homogeneous population including one or more recombinant RSV Fproteins that are, for example, all stabilized in a prefusionconformation. In some embodiments, in the homogeneous population atleast about 90% of the recombinant RSV F proteins (such as at leastabout 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% of the RSV Fproteins) are stabilized in the prefusion conformation. In someembodiments, in the homogeneous population, at least about 90% of therecombinant RSV F proteins (such as at least about 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 99.9% of the RSV F proteins) are specificallybound by a prefusion specific antibody (e.g., D25 or AM22 antibody),and/or include a RSV F prefusion specific conformation (such asantigenic site Ø). It will be understood that a homogeneous populationof RSV F proteins in a particular conformation can include variations(such as protein modification variations, e.g., glycosylation state),that do not alter the conformational state of the RSV F protein. Inseveral embodiments, the population of recombinant RSV F protein remainshomogeneous over time. For example, the PreF antigen can include arecombinant RSV F protein that, when dissolved in aqueous solution,forms a population of recombinant RSV F proteins that is stabilized in aprefusion conformation for at least 12 hours, such as at least 24 hours,at least 48 hours, at least one week, at least two weeks, or more.

In several embodiments, the isolated PreF antigens are substantiallyseparated from RSV F proteins in a post-fusion conformation. Thus, thePreF antigen can be, for example, at least 80% isolated, at least 90%,95%, 98%, 99%, or even 99.9% separated from RSV F proteins in apostfusion conformation. In several embodiments, the PreF antigens arealso separated from RSV F proteins that do not include antigen site Øand/or are not specifically bound by a prefusion specific monoclonalantibody (such as D25 or AM22). For example, the PreF antigen can be atleast 80% isolated, at least 90%, 95%, 98%, 99%, or even 99.9% separatedfrom RSV F proteins that do not include antigen site Ø and/or are notspecifically bound by a prefusion specific monoclonal antibody (such asD25 or AM22).

In some embodiments, the PreF antigens are provided as a homogenouspopulation that does not include detectable RSV F protein in apost-fusion conformation. RSV F protein is detectable by negative stainelectron microscope and/or specific binding by a postfusion antibody.

1. Recombinant RSV F Proteins Stabilized in a Prefusion Conformation

The PreF antigens disclosed herein can include a recombinant RSV Fprotein stabilized in a prefusion conformation and include an F₁polypeptide and a F₂ polypeptide. The F₁ polypeptide, F₂ polypeptide, orboth, can include at least one modification (e.g., an amino acidsubstitution) that stabilizes the recombinant RSV F protein in itsprefusion conformation. Stabilization of the recombinant RSV F proteinin the prefusion conformation preserves at least one prefusion-specificepitope (i.e., an epitope present in the pre- (but not post-) fusionconformation of the RSV F protein) that specifically binds to a RSV Fprefusion-specific monoclonal antibody (i.e., an antibody thatspecifically binds to the RSV F protein in a prefusion conformation, butnot a post fusion conformation). Thus, the disclosed PreF antigens arespecifically bound by a prefusion specific antibody (e.g., D25 or AM22antibody), and/or includes a RSV F prefusion specific conformation (suchas antigenic site Ø).

In some examples, the PreF antigen includes a recombinant RSV F proteinincluding a F₁ and/or F₂ polypeptide from a RSV A virus, for example,for example, a F₁ and/or F₂ polypeptide from a RSV F₀ protein providedas one of SEQ ID NOs: 1-128. In some examples, the PreF antigen includesa recombinant RSV F protein including a F₁ and/or F₂ polypeptide from aRSV B virus, for example, for example, a F₁ and/or F₂ polypeptide from aRSV F₀ protein provided as one of SEQ ID NOs: 129-177. In some examples,the PreF antigen includes a recombinant RSV F protein including a F₁and/or F₂ polypeptide from a RSV bovine virus, for example, for example,a F₁ and/or F₂ polypeptide from a RSV F₀ protein provided as one of SEQID NOs: 178-184. The person of ordinary skill in the art will appreciatethat F₁ and/or F₂ polypeptides from other RSV subtypes can also be used.The person of ordinary skill in the art will appreciate that therecombinant RSV F protein can include modifications of the native RSVsequences, such as amino acid substitutions, deletions or insertions,glycosylation and/or covalent linkage to unrelated proteins, as long asthe PreF antigen retains the recombinant RSV F protein stabilized in aprefusion conformation. RSV F proteins from the different RSV groups, aswell as nucleic acid sequences encoding such proteins and methods forthe manipulation and insertion of such nucleic acid sequences intovectors, are disclosed herein and known in the art (see, e.g., Tan etal., PLOS one, 7: e51439, 2011; Sambrook et al., Molecular Cloning, aLaboratory Manual, 2d edition, Cold Spring Harbor Press, Cold SpringHarbor, N.Y. (1989); Ausubel et al., Current Protocols in MolecularBiology, Greene Publishing Associates and John Wiley & Sons, New York,N.Y. (1994)).

In some examples, the PreF antigen includes a recombinant RSV F proteinincluding a F₁ and/or F₂ polypeptide including a polypeptide sequenceshaving at least 75% (for example at least 85%, 90%, 95%, 96%, 97%, 98%or 99%) sequence identity with a RSV F₁ and/or F₂ polypeptide from a RSVA virus, for example, a F₁ and/or F₂ polypeptide from a RSV F₀ proteinprovided as one of SEQ ID NOs: 1-128. In further examples, the PreFantigen includes a recombinant RSV F protein including a F₁ and/or F₂polypeptide including a polypeptide sequences having at least 75% (forexample at least 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identitywith a RSV F₁ and/or F₂ polypeptide from a RSV B virus, for example, aF₁ and/or F₂ polypeptide from a RSV F₀ protein provided as one of SEQ IDNOs: 129-177. In further examples, the PreF antigen includes arecombinant RSV F protein including a F₁ and/or F₂ polypeptide includinga polypeptide sequences having at least 75% (for example at least 85%,90%, 95%, 96%, 97%, 98% or 99%) sequence identity with a RSV F₁ and/orF₂ polypeptide from a RSV bovine virus, for example, a F₁ and/or F₂polypeptide from a RSV F₀ protein provided as one of SEQ ID NOs:178-184.

In several embodiments, the PreF antigen includes a recombinant RSV Fprotein including a F₁ polypeptide including or consisting of at least300 consecutive amino acids (such as at least 310, 320, 330, 340, 350,360, 370, 380, 390, 400, 410, 420, or 430 consecutive amino acids) froma native F₁ polypeptide sequence, such as positions 137-513 of one ofSEQ ID NOs: 1-184, including any polypeptide sequences having at least75% (for example at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequenceidentity to a native F₁ polypeptide sequence, such as positions 137-513of any one of SEQ ID NOs: 1-184. For example, in some embodiments, thePreF antigen includes a recombinant F protein includes a F₁ polypeptideincluding or consisting of positions 137-513, 137-481, 137-491, orposition 137 to the C-terminus, or positions 137-to the transmembranedomain, of any one of SEQ ID NOs: 1-184, including any polypeptidesequences having at least 75% (for example at least 85%, 90%, 95%, 96%,97%, 98% or 99%) sequence identity to a native F₁ polypeptide sequence,such as positions 137-513, or position 137 to the C-terminus, orpositions 137-to the transmembrane domain, any one of SEQ ID NOs: 1-184.The person of ordinary skill in the art will appreciate that the PreFantigen including the recombinant RSV F protein can include a F1polypeptide with N- or C-terminal truncations (for example, deletion of1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 or moreamino acids) compared to extracellular region of a native F1 polypeptide(for example, positions 137-524), as long as the PreF antigen isspecifically bound by a prefusion specific antibody (e.g., D25 or AM22antibody), and/or includes a RSV F prefusion specific conformation (suchas antigenic site Ø).

In some embodiments, the PreF antigen includes a F₁ polypeptideincluding a maximum length, for example no more than 300, 310, 320, 330,340, 350, 360, 370, 380, 390, 400, 410, 420, 430, or no more than 440amino acids in length. The F₁ polypeptide may include, consist orconsist essentially of the disclosed sequences. The disclosed contiguousF₁ polypeptide sequences may also be joined at either end to otherunrelated sequences (for examiner, non-RSV F₁ protein sequences, non-RSVF protein sequences, non-RSV, non-viral envelope, or non-viral proteinsequences)

In several embodiments, the PreF antigen includes a recombinant RSV Fprotein including a F₂ polypeptide including or consisting of at least60 consecutive amino acids (such as at least 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,102, 103, 104, 105, 106, 107, 108 or 109 consecutive amino acids) from anative F₂ polypeptide sequence, such as positions 26-109 of any one ofSEQ ID NOs: 1-184, including a polypeptide sequences having at least 75%(for example at least 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequenceidentity to a native F₁ polypeptide sequence, such as positions 26-109any one of SEQ ID NOs: 1-184. For example, in some embodiments, the PreFantigen includes a recombinant F protein including a F₂ polypeptideincluding or consisting of 70-109 consecutive amino acids (such as60-100, 75-95, 80-90, 75-85, 80-95, 81-89, 82-88, 83-87, 83-84, or 84-85consecutive amino acids) from a native F₂ polypeptide sequence, such aspositions 26-109 any one of SEQ ID NOs: 1-184, including any polypeptidesequences having at least 75% (for example at least 85%, 90%, 95%, 96%,97%, 98% or 99%) sequence identity to a native F₂ polypeptide sequence,such as positions 137-513 any one of SEQ ID NOs: 1-184.

In some embodiments, the PreF antigen includes a F₂ polypeptide that isalso of a maximum length, for example no more than 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or100 amino acids in length. The F₂ polypeptide may include, consist orconsist essentially of the disclosed sequences. The disclosed contiguousF₂ polypeptide sequences may also be joined at either end to otherunrelated sequences (for examiner, non-RSV F₂ protein sequences, non-RSVF protein sequences, non-RSV, non-viral envelope, or non-viral proteinsequences).

In some embodiments, the PreF antigen includes a recombinant RSV Fprotein including a F₂ polypeptide including or consisting of at least60 consecutive amino acids (such as at least 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,102, 103, 104, 105, 106, 107, 108 or 109 consecutive amino acids) from anative F₂ polypeptide sequence, such as positions 26-109 of any one ofSEQ ID NOs: 1-184, including polypeptide sequences having at least 75%(for example at least 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequenceidentity to a native F₂ polypeptide sequence, such as amino acids 26-109any one of SEQ ID NOs: 1-184, and further includes a F₁ polypeptideincluding or consisting of at least 300 consecutive amino acids (such asat least 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, or430 consecutive amino acids) from a native F₁ polypeptide sequence, suchas positions 137-513 of one of SEQ ID NOs: 1-184, including anypolypeptide sequences having at least 75% (for example at least 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99%) sequence identity to a native F₁ polypeptidesequence, such as positions 137-513 of any one of SEQ ID NOs: 1-184.

In one non-limiting example, the PreF antigen includes a recombinant RSVF protein including a F₂ polypeptide and a F₁ polypeptide includingpositions 26-109 and 137-513, respectively, of any one of SEQ ID NOs:1-184, including polypeptide sequences having at least 75% (for exampleat least 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to apositions 26-109 and 137-513, respectively, of any one of SEQ ID NOs:1-184.

As noted above, the RSV F protein is initially synthesized as a F₀precursor protein and is cleaved at multiple sites (including twoconserved furin cleavage sites) during maturation. Thus, the native RSVF protein lacks the N-terminal signal peptide and the pep27 peptide (ora portion thereof) of the F₀ precursor protein. In several embodiments,the disclosed recombinant RSV F proteins stabilized in the prefusionconformation do not include the signal peptide (or a portion thereof)and/or do not include the pep27 peptide (or a portion thereof). Theperson of ordinary skill in the art will appreciate that recombinant RSVF proteins lacking the RSV F signal peptide and/or pep27 peptide can begenerated by expressing the recombinant F₀ polypeptide in cells wherethe signal peptide and the pep27 peptide will be excised from the F₀precursor by cellular proteases.

Several embodiments include a PreF antigen including a multimer of anyof the disclosed recombinant RSV F proteins, for example, a multimerincluding 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more of the disclosedrecombinant RSV F proteins. In several examples, any of the disclosedrecombinant RSV F proteins can be linked (e.g., via a peptide linker) toanother of the recombinant RSV F proteins to form the multimer.

It is understood in the art that some variations can be made in theamino acid sequence of a protein without affecting the activity of theprotein. Such variations include insertion of amino acid residues,deletions of amino acid residues, and substitutions of amino acidresidues. These variations in sequence can be naturally occurringvariations or they can be engineered through the use of geneticengineering technique known to those skilled in the art. Examples ofsuch techniques are found in Sambrook J, Fritsch E F, Maniatis T et al.,in Molecular Cloning—A Laboratory Manual, 2nd Edition, Cold SpringHarbor Laboratory Press, 1989, pp. 9.31-9.57), or in Current Protocolsin Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, bothof which are incorporated herein by reference in their entirety. Thus,in some embodiments, the PreF antigen includes a F₁ polypeptide, a F₂polypeptide, or both a F₁ and F₂ polypeptide, that include one or moreamino acid substitutions compared to the corresponding native RSVsequence. For example, in some embodiments, the F₁ polypeptide, F₂polypeptide, or both the F₁ polypeptide and the F₂ polypeptide, includeup to 20 (such as up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, or 19) amino acid substitutions compared to a native F₁polypeptide sequence, such as a native RSV sequence set forth as any oneof SEQ ID NOs: 1-184, wherein the PreF antigen is specifically bound bya RSV F prefusion specific antibody (e.g., D25 or AM22 antibody), and/orincludes a RSV F prefusion specific conformation (such as antigenic siteØ). For example, in some embodiments, the PreF antigen includes arecombinant RSV F protein in a prefusion conformation that is modifiedto increase expression of the protein for protein productions purposes,e.g., by elimination of one or more nuclear localization signals presenton the RSV F protein. Manipulation of the nucleotide sequence encodingthe F₁ or F₂ polypeptide sequence (such as a nucleotide sequenceencoding the F₀ polypeptide including the F₁ and F₂ polypeptides) usingstandard procedures, including in one specific, non-limiting,embodiment, site-directed mutagenesis or in another specific,non-limiting, embodiment, PCR, can be used to produce such variants.Alternatively, the F₁ and F₂ polypeptides can be synthesized usingstandard methods. The simplest modifications involve the substitution ofone or more amino acids for amino acids having similar biochemicalproperties. These so-called conservative substitutions are likely tohave minimal impact on the activity of the resultant protein.

a. Prefusion Stabilizing Modifications

As disclosed herein, the RSV F protein undergoes a structuralrearrangement between its pre- and post-fusion conformations. As shownin FIG. 2B, the N-terminal region of the F₁ polypeptide in the prefusionconformation (corresponding in part to the membrane distal lobe shown inFIG. 2A) includes the indicated α2, α3, β3, β4, and α4 helical and betasheet structures, whereas the corresponding region of the N-terminus ofthe F₁ polypeptide in the postfusion structure includes an extended α5helical structure—the α2, α3, β3, β4, and α4 helical and beta sheetstructures are absent. Further, the C-terminal region of the F₁polypeptide in the prefusion conformation (corresponding in part to themembrane proximal lobe shown in FIG. 2A) includes the indicated β22, α9,and β23 beta sheet and helical structures, whereas the correspondingC-terminal region of the F₁ polypeptide in the postfusion conformationstructure includes an extended α10 helical structure and extendedcoil—the β22, α9, and β23 beta sheet and helical structures are absent.Thus, the membrane distal and membrane proximal lobes of the RSV Fprotein in its prefusion conformation include several distinctstructural elements that are absent from the corresponding regions ofthe RSV F protein in its postfusion conformation.

Guided by the structural features identified in the pre- and post-fusionconformations of the RSV F protein, several modes of stabilizing the RSVF protein in a prefusion conformation are available, including aminoacid substitutions that introduce one or more disulfide bonds, fillcavities within the RSV F protein, alter the packing of residues in theRSV F protein, introduce N-linked glycosylation sites, and combinationsthereof.

The stabilizing modifications provided herein are targeted modificationsthat stabilize the recombinant RSV F protein in the prefusionconformation. Thus, in several embodiments, the RSV F protein is notstabilized by non-specific cross-linking, such as glutaraldehydecrosslinking, for example glutaraldehyde crosslinking of membrane boundRSV F trimers.

In some non-limiting embodiments, the PreF antigen includes arecombinant RSV F protein stabilized in a prefusion conformation byintroduction of a disulfide bond, wherein the recombinant RSV F proteinincludes S155C and S290C; G151C and I288C; A153C and K461C; A149C andY458C; G143C and S404S substitutions; or Y33C and V469C amino acidsubstitutions. Non-limiting examples of precursor proteins of suchrecombinant RSV F proteins (including a Foldon domain linked to theC-terminus of the F1 polypeptide) are set forth herein as SEQ ID NO:185, SEQ ID NO: 189, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, andSEQ ID NO: 211.

i. Disulfide Bonds

In several embodiments, the PreF antigen includes a recombinant RSV Fprotein stabilized in a prefusion conformation by at least one disulfidebond including a pair of cross-linked cysteine residues. For example, insome embodiments, any of the disclosed recombinant RSV F protein can bestabilized in a prefusion conformation by any one of 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 disulfide bonds including a pair of cross-linked cysteineresidues. In one specific non-limiting example, the recombinant RSV Fprotein is stabilized in a prefusion conformation by a single pair ofcross-linked cysteine residues. In another non-limiting example, any ofthe disclosed recombinant RSV F protein is stabilized in a prefusionconformation by two pairs of crosslinked cysteine residues.

The cysteine residues that form the disulfide bond can be introducedinto native RSV F protein sequence by one or more amino acidsubstitutions. For example, in some embodiments, a single amino acidsubstitution introduces a cysteine that forms a disulfide bond with acysteine residue present in the native RSV F protein sequence. Inadditional embodiments, two cysteine residues are introduced into anative RSV sequence to form the disulfide bond. The location of thecysteine (or cysteines) of a disulfide bond to stabilize the RSV Fprotein in a prefusion conformation can readily be determined by theperson of ordinary skill in the art using the disclosed structure of RSVF protein in its prefusion conformation, and the previously identifiedstructure of RSV F protein in its post fusion conformation.

For example, the amino acid positions of the cysteines are typicallywithin a sufficiently close distance for formation of a disulfide bondin the prefusion conformation of the RSV F protein. Methods of usingthree-dimensional structure data to determine if two residues are withina sufficiently close distance to one another for disulfide bondformation are known (see, e.g., Peterson et al., Protein engineering,12:535-548, 1999 and Dombkowski, Bioinformatics, 19:1852-1853, 3002(disclosing DISULFIDE BY DESIGN™), each of which is incorporated byreference herein). For example, residues can be selected manually, basedon the three dimensional structure of RSV F protein in a prefusionconformation provided herein, or a software, such as DISULFIDEBYDESIGN™,can be used. Without being bound by theory, ideal distances forformation of a disulfide bond are generally considered to be about ˜5.6Å for Cα-Cα distance, ˜2.02 Å for Sγ-Sγ distance, and 3.5-4.25 Å forCβ-Cβ distance. The person of ordinary skill in the art will appreciatethat variations from these distances are included when selectingresidues in a three dimensional structure that can be substituted forcysteines for introduction of a disulfide bond. For example, in someembodiments the selected residues have a Cα-Cα distance of less than 7.0Å and/or a Cβ-Cβ distance of less than 4.7 Å. In some embodiments theselected residues have a Cα-Cα distance of from 2.0-8.0 Å and/or a Cβ-Cβdistance of from 2.0-5.5 Å. In several embodiments, the amino acidpositions of the cysteines are within a sufficiently close distance forformation of a disulfide bond in the prefusion, but not post-fusion,conformation of the RSV F protein.

The person of ordinary skill in the art can readily determine therelative position of a particular amino acid between the pre- andpost-fusion conformations of the RSV F protein, for example by comparingthe prefusion structures defined herein by the structural coordinatesprovided in Table 1, with the previously identified postfusion structuredescribed in McLellan et al., J. Virol., 85, 7788, 2011, with structuralcoordinates deposited as PDB Accession No. 3RRR). Methods of determiningrelative position of a particular amino acid between the two proteinstructures (e.g., between the three dimensional structures pre- andpost-fusion RSV F protein) are known. For example the person of ordinaryskill in the art can use known superimposition methods to compare thetwo structures (e.g., methods using the LSQKAB program (Kabsch W. Acta.Cryst. A32 922-923 (1976)). In one example, the pre- and postfusionstructures can be superimposed by using LSQKAB to align F proteinpositions 26-60, 77-97, 220-322, and 332-459 defined by the structuralcoordinates provided in Table 1, with the F protein positions 26-60,77-97, 220-322, and 332-459 defined by the structural coordinatesdeposited as PDB Accession No. 3RRR, and comparing the distance betweenthe Cα atom for each residue in the pre- and post-fusion structures toidentify the deviation of particular residues between the twostructures.

In several embodiments, the PreF antigen includes a recombinant RSV Fprotein stabilized in a prefusion conformation by a disulfide bondbetween a cysteine introduced into an amino acid position that changesconformation, and a cysteine introduced into an amino acid position thatdoes not change conformation, between the pre- and post-fusionstructures, respectively. For example, in some embodiments, the PreFantigen includes a recombinant RSV F protein including amino acidsubstitutions introducing a pair of cysteines, wherein the firstcysteine is in an amino acid position of the RSV F protein that has aroot mean square deviation of at least 5 (such as at least 6, at least7, at least 8, at least 9 or at least 10) angstroms between thethree-dimensional structure of the RSV F protein pre- and post-fusionconformations, and the second cysteine is in an amino acid position ofthe RSV F protein that has a root mean square deviation of less than 4(such as less than 3, 2, or 1) angstroms between the three-dimensionalstructure of the RSV F protein pre- and post-fusion conformations,wherein the PreF antigen is specifically bound by a prefusion specificantibody (e.g., D25 or AM22 antibody), and/or includes a RSV F prefusionspecific conformation (such as antigenic site Ø).

Based on a comparison of the pre- and post-fusion RSV F structures,there are at least two regions that undergo large conformationalchanges, located at the N- and C-termini of the F₁ subunit (residues137-216 and 461-513, respectively). For example, as illustrated in FIG.2B, the positions 137-216 and 461-513 of the F₁ polypeptide undergostructural rearrangement between the Pre-and Post-F proteinconformations, whereas positions 217-460 of the F₁ polypeptide remainrelatively unchanged. Thus, in some embodiments, the PreF antigenincludes a recombinant RSV F protein stabilized in a prefusionconformation by a disulfide bond between a first cysteine in one ofpositions 137-216 or 461-513 of the F₁ polypeptide, and a secondcysteine in one of positions 217-460 of the F₁ polypeptide. In furtherembodiments, the PreF antigen includes a recombinant RSV F proteinstabilized in a prefusion conformation by a disulfide bond between afirst cysteine in one of positions 137-216 or 461-513 of the F₁polypeptide, and a second cysteine in a position of the F₂ polypeptide,such as one of positions 26-109 (for example, one of positions 26-61 or77-97) of the F₂ polypeptide.

In additional embodiments, the PreF antigen includes a recombinant RSV Fprotein stabilized in a prefusion conformation by a disulfide bondbetween cysteines that are introduced into amino acid positions thatchange conformation between the pre- and post-fusion structures,respectively. For example, in some embodiments, the PreF antigenincludes a recombinant RSV F protein including amino acid substitutionsintroducing a pair of cysteines, wherein the first cysteine and thesecond cysteine is in an amino acid position of the RSV F protein thathas a root mean square deviation of at least 5 (such as at least 6, atleast 7, at least 8, at least 9 or at least 10) angstroms between thethree-dimensional structure of the RSV F protein pre- and post-fusionconformations, wherein the PreF antigen includes specific bindingactivity to an RSV F prefusion specific antibody (e.g., D25 or AM22antibody), and/or includes a RSV F prefusion specific epitope (e.g., aD25 or AM22 epitope). In some such embodiments, the PreF antigenincludes a recombinant RSV F protein stabilized in a prefusionconformation by a disulfide bond between a the first cysteine and thesecond cysteine in positions 137-216 of the F₁ polypeptide. Inadditional embodiments, the PreF antigen includes a recombinant RSV Fprotein stabilized in a prefusion conformation by a disulfide bondbetween a the first cysteine and the second cysteine in positions461-513 of the F₁ polypeptide. In further embodiments, the PreF antigenincludes a recombinant RSV F protein stabilized in a prefusionconformation by a disulfide bond between a the first cysteine and thesecond cysteine in positions 137-216 and 461-513, respectively, of theF₁ polypeptide.

The person of ordinary skill in the art can readily determine thelocation of a particular amino acid in the pre- and post-fusionconformations of the RSV F protein (and any difference in a positionbetween the two conformations) using the structural coordinates of thethree-dimensional structure the RSV F protein in the pre fusionconformation are set forth in Table 1, and the structural coordinates ofthe three-dimensional structure of the RSV F protein in the postfusionconformation are set forth in Protein Databank Accession No. 3RRR. Forexample, such comparison methods are described in Example 1, below.Table 5 provides examples of cysteine pairs and amino acid substitutionsthat can be used to stabilize a RSV F protein in a prefusionconformation.

TABLE 5 Exemplary Cysteine Pairs for Disulfide Bond Stabilization Fprotein Residue Pair(s) for Cysteine Substitutions corresponding SEQ IDSubstitution to SEQ ID NO: 124 NO F₁ substitutions-Intra-ProtomerDisulfide Bond 1 155 and 290 S155C and S290C 185 2 151 and 288 G151C andI288C 189 3 137 and 337 F137C and T337C 213 4 397 and 487 T397C andE487C 247 5 138 and 353 L138C and P353C 257 6 341 and 352 W341C andF352C 267 7 403 and 420 S403C and T420C 268 8 319 and 413 S319C andI413C 269 9 401 and 417 D401C and Y417C 270 10 381 and 388 L381C andN388C 271 11 320 and 415 P320C and S415C 272 12 319 and 415 S319C andS415C 273 13 331 and 401 N331C and D401C 274 14 320 and 335 P320C andT335C 275 15 406 and 413 V406C and I413C 277 16 381 and 391 L381C andY391C 278 17 357 and 371 T357C and N371C 279 18 403 and 417 S403C andY417C 280 19 321 and 334 L321C and L334C 281 20 338 and 394 D338C andK394C 282 21 288 and 300 I288C and V300C 284 F₂ andF₁Substitutions-Intra-Protomer Disulfide Bond 22 60 and 194 E60C andD194C 190 23 33 and 469 Y33C and V469C 211 24 54 and 154 T54C and V154C212 25 59 and 192 I59C and V192C 246 26 46 and 311 S46C and T311C 276 2748 and 308 L48C and V308C 283 28 30 and 410 E30C and L410C 285 F₁substitutions-Inter-Protomer Disulfide Bond 29 400 and 489 T400C andD489C 201 30 144 and 406 V144C and V406C 202 31 153 and 461 A153C andK461C 205 32 149 and 458 A149C and Y458C 207 33 143 and 404 G143C andS404S 209 34 346 and 454 S346C and N454C 244 35 399 and 494 K399C andQ494C 245 36 146 and 407 S146C and I407C 264 37 374 and 454 T374C andN454C 265 38 369 and 455 T369C and T455C 266 39 402 and 141 V402C andL141C 302 F₂ and F₁ Substitutions-Inter-Protomer Disulfide Bond 40 74and 218 A74C and E218C 243 Amino acid insertions to Orient the Disulfidebond 41 145 and 460 (Inter), AA insertion between S145C and 460C; AAinsertion between 338 positions 146 and 147 positions 146/147 42 183 and423 (Inter), AAA insertion between N183C and K423C; AAA insertion 339positions 182 and 183 between positions 182/183 43 330 and 430 (Inter);CAA insertion between A329C and S430C; and a CAA insertion 340 positions329 and 330 between positions 329 and 330 Combinations 44 155 and 290(Intra); and 402 and 141 (Inter) S155C and S290C; and V402C and 303L141C 45 155 and 290(Intra); and 74 and 218 S155C and S290C; and A74Cand E218C 263 46 155 and 290 (Intra); and 146 and 460 (Inter); S155C andS290C; and S146C and 258 G insertion between position 460 and 461 N460C;G insertion between position 460 and 461 47 155 and 290 (Intra); and 345and 454(Inter); S155C and S290C; and N345C and 259 C insertion betweenpositions 453 and 454 N454G; C insertion between positions 453 and 45448 155 and 290 (Intra); and 374 and 454(Inter); S155C and S290C; andT374C and 260 C insertion between positions 453 and 454 N454G; Cinsertion between positions 453 and 454 49 155 and 290 (Intra); and 239and 279(Inter); S155C and S290C; and S238G and 261 C insertion betweenpositions 238 and 239 Q279C; C insertion between positions 238 and 23950 155 and 290 (Intra); and 493 paired with C S155C and S290C; and S493Cpaired 262 insertion between positions 329 and 330 with a C insertionbetween positions 329 and 330 51 183 and 428 (Inter), G insertionbetween N183C and N428C; G insertion between 296 positions 182 and 183positions 182 and 183 52 183 and 428 (Inter), C insertion between N183Cand N427G; C insertion between 297 positions 427 and 428 positions 427and 428 53 155 and 290 (Intra); and 183 and 428(Inter); S155C and S290C;and N183C and 298 G insertion between positions 182 and 183 N428C; Ginsertion between positions 182 and 183 54 155 and 290 (Intra); and 183and 428(Inter); S155C and S290C; and N183C and 299 C insertion betweenpositions 427 and 428 N427G; C insertion between positions 427 and 428

In some embodiments, the PreF antigen includes a recombinant RSV Fprotein including one or more (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10)disulfide bonds, including disulfide bond between cysteine residueslocated at the RSV F positions listed in one or more of rows 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, or 51 of column 2 of Table 5,wherein the PreF antigen is specifically bound by a prefusion specificantibody (e.g., D25 or AM22 antibody), and/or includes a RSV F prefusionspecific conformation (such as antigenic site Ø).

In further embodiments, the PreF antigen includes a recombinant RSV Fprotein including one or more (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10)disulfide bonds, including disulfide bonds between cysteine residuesthat are introduced by the cysteine amino acid substitutions listed inone or more of rows 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or51 of column 3 of Table 5, wherein the PreF antigen is specificallybound by a prefusion specific antibody (e.g., D25 or AM22 antibody),and/or includes a RSV F prefusion specific conformation (such asantigenic site Ø).

The SEQ ID NOs listed in column 4 of Table 5 set forth amino acidsequences including the indicated substitutions, as well as, a signalpeptide, F₂ polypeptide (positions 26-109), a pep27 polypeptide(positions 27-136), a F₁ polypeptide (positions 137-513), atrimerization domain (a Foldon domain) and a thrombin cleavage site(LVPRGS) and purification tags (his-tag (HHHHHH) and Strep Tag II(SAWSHPQFEK)).

Thus, in additional embodiments, the PreF antigen includes a recombinantRSV F protein including a F₁ polypeptide and a F₂ polypeptide as setforth in any one of the SEQ ID NOs listed in column 4 of Table 5, suchas a SEQ ID NO listed in one of rows 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, or 51 of column 4 of Table 5, wherein the PreF antigen isspecifically bound by a prefusion specific antibody (e.g., D25 or AM22antibody), and/or includes a RSV F prefusion specific conformation (suchas antigenic site Ø). In further embodiments, the PreF antigen includesa RSV F protein including a F₁ polypeptide and a F₂ polypeptide, whereinthe F₂ and the F₁ polypeptide include the amino acid sequence set forthas positions 26-109 and 137-513, respectively, of any one of the SEQ IDNOs listed in column 4 of Table 5, such as a SEQ ID NO listed in one ofrows 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 51 of column 4 ofTable 5, wherein the PreF antigen is specifically bound by a prefusionspecific antibody (e.g., D25 or AM22 antibody), and/or includes a RSV Fprefusion specific conformation (such as antigenic site Ø).

In further embodiments, the PreF antigen includes a recombinant RSV Fprotein including one or more (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10)intra-protomer disulfide bonds, including a disulfide bond betweencysteine residues located at the RSV F positions of the F₁ polypeptidelisted in of one or more of rows 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, or 21 of column 2 Table 5. In furtherembodiments, the PreF antigen includes a recombinant RSV F proteinincluding one or more (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10)intra-protomer disulfide bonds, including disulfide bonds betweencysteine residues that are introduced by the F₁ polypeptide amino acidsubstitutions listed in of one or more of rows 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 of column 3 ofTable 5. In any of these embodiments, the PreF antigen is specificallybound by a prefusion specific antibody (e.g., D25 or AM22 antibody),and/or includes a RSV F prefusion specific conformation (such asantigenic site Ø).

In further embodiments, the PreF antigen includes a recombinant RSV Fprotein including one or more (such as 2, 3, 4, 5, 6, or 7)intra-protomer disulfide bonds, including a disulfide bond betweencysteine residues located at the RSV F positions of the F₂ and F₁polypeptides listed in of one or more of rows 22, 23, 24, 25, 26, 27, or28 of column 2 of Table 5. In further embodiments, the PreF antigenincludes a recombinant RSV F protein including one or more (such as 2,3, 4, 5, 6, or 7) intra-protomer disulfide bonds, including disulfidebond between cysteine residues that are introduced by the F₂ and F₁polypeptide amino acid substitutions listed in of one or more of rows22, 23, 24, 25, 26, 27, or 28 of column 3 of Table 5. In any of theseembodiments, the PreF antigen is specifically bound by a prefusionspecific antibody (e.g., D25 or AM22 antibody), and/or includes a RSV Fprefusion specific conformation (such as antigenic site Ø).

In further embodiments, the PreF antigen includes a recombinant RSV Fprotein including one or more (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10)inter-protomer disulfide bonds, including a disulfide bond betweencysteine residues located at the RSV F positions of the F₁ polypeptidelisted in one or more of rows 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or39 of column 2 of Table 5. In further embodiments, the PreF antigenincludes a recombinant RSV F protein including one or more (such as 2,3, 4, 5, 6, 7, 8, 9 or 10) inter-protomer disulfide bonds, includingdisulfide bond between cysteine residues that are introduced by the F₁polypeptide amino acid substitutions listed in of one or more of rows29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 of column 3 of Table 5. Inany of these embodiments, the PreF antigen is specifically bound by aprefusion specific antibody (e.g., D25 or AM22 antibody), and/orincludes a RSV F prefusion specific conformation (such as antigenic siteØ).

In further embodiments, the PreF antigen includes a recombinant RSV Fprotein including an inter-protomer disulfide bond between cysteineresidues located at the RSV F positions of the F₂ and F₁ polypeptideslisted in column 2 of row 40 of Table 5. In further embodiments, thePreF antigen includes a recombinant RSV F protein including aninter-protomer disulfide bond between cysteine residues that areintroduced by the amino acid substitutions in the F₂ and F₁ polypeptidelisted in column 3 of row 40 of Table 5. In any of these embodiments,the PreF antigen is specifically bound by a prefusion specific antibody(e.g., D25 or AM22 antibody), and/or includes a RSV F prefusion specificconformation (such as antigenic site Ø).

In some embodiments, amino acids can be inserted (or deleted) from the Fprotein sequence to adjust the alignment of residues in the F proteinstructure, such that particular residue pairs are within a sufficientlyclose distance to form an intra- or inter-protomer disulfide bond in theprefusion, but not postfusion, conformation. In several suchembodiments, the PreF antigen includes a recombinant RSV F proteinincluding a disulfide bond between cysteine residues located at the RSVF positions of the F₁ polypeptide, as well as the amino acid insertion,listed in one or more of rows 41, 42, or, 43 of column 2 of Table 5. Infurther embodiments, the PreF antigen includes a recombinant RSV Fprotein including a disulfide bond between cysteine residues that areintroduced by the F₁ polypeptide amino acid substitutions, as well asthe amino acid insertion, listed in of one or more of rows 41, 42, or,43 of column 3 of Table 5.

In one example, the PreF antigen includes a recombinant RSV F proteinstabilized in a prefusion conformation that includes a disulfide bondbetween cysteines at F1 positions 155 and 290, such as a recombinant F1polypeptide protein with S155C and S290C substitutions.

In some embodiments, the PreF antigen includes a recombinant RSV Fprotein including a combination of two or more of the disulfide bondsbetween cysteine residues listed above, wherein the PreF antigen isspecifically bound by a prefusion specific antibody (e.g., D25 or AM22antibody), and/or includes a RSV F prefusion specific conformation (suchas antigenic site Ø). It is understood that some combinations will notresult in a RSV F protein stabilized in a prefusion conformation; suchcombinations can be identified by methods disclosed herein, for exampleby confirming that the antigen containing such a polypeptide isspecifically bound by a prefusion specific antibody (e.g., D25 or AM22antibody), and/or includes a RSV F prefusion specific conformation (suchas antigenic site Ø)

ii. Cavity Filling Amino Acid Substitutions

Comparison of the structure of the prefusion conformation of the RSV Fprotein (e.g., in complex with D25 Fab as disclosed herein) to thestructure of the postfusion RSV F protein (disclosed, e.g., in asdisclosed in McLellan et al., J. Virol., 85, 7788, 2011) identifiesseveral internal cavities or pockets in the prefusion conformation thatmust collapse for F to transition to the postfusion conformation. Thesecavities include those listed in Table 6, below.

Accordingly, in several embodiments, the PreF antigen includes arecombinant RSV F protein stabilized in a prefusion conformation by oneor more amino acid substitutions that introduce an amino acid thatreduces the volume of an internal cavity that collapses in thepostfusion conformation of RSV F protein. For example, cavities arefilled by substituting amino acids with large side chains for those withsmall side chains. The cavities can be intra-protomer cavities, orinter-protomer cavities. One example of a RSV F cavity filling aminoacid substitution to stabilize the RSV protein in its prefusionconformation a RSV F protein with S190F and V207L substitutions.

The person of ordinary skill in the art can use methods provided hereinto compare the structures of the pre- and post-fusion conformations ofthe RSV F protein to identify suitable cavities, and amino acidsubstitutions for filling the identified cavities. Exemplary cavitiesand amino acid substitutions for reducing the volume of these cavitiesare provided in Table 6, below.

TABLE 6 Exemplarity cavity-filling amino acid substitution RowCavity/Cavities A.A. Substitutions SEQ ID NO: 1 Ser190 190F and 207L 1912 Val207 207L and 220L 193 3 Ser190 and Val296 296F and 190F 196 4Ala153 and Val207 220L and 153W 197 5 Val207 203W 248 6 Ser190 andVa1207 83W and 260W 192 7 Val296 58W and 298L 195 8 Val90 87F and 90L194

The indicated cavities are referred to by a small residue abutting thecavity that can be mutated to a larger residue to fill the cavity. Itwill be understood that other residues (besides the one the cavity isnamed after) could also be mutated to fill the same cavity.

Thus, in some embodiments, the PreF antigen includes a recombinant RSV Fprotein including one or more amino acid substitutions that reduce thevolume of one or more of the cavities listed in column 2 of Table 6,wherein the PreF antigen is specifically bound by a prefusion specificantibody (e.g., D25 or AM22 antibody), and/or includes a RSV F prefusionspecific conformation (such as antigenic site Ø). In additionalembodiments, the PreF antigen includes a recombinant RSV F proteinincluding one or more of the amino acid substitutions listed in of row1, 2, 3, 4, 5, 6, 7, or 8 of column 3 of Table 6, wherein the PreFantigen is specifically bound by a prefusion specific antibody (e.g.,D25 or AM22 antibody), and/or includes a RSV F prefusion specificconformation (such as antigenic site Ø).

The SEQ ID NOs listed in Table 6 set forth amino acid sequencesincluding the indicated substitutions, as well as, a signal peptide, F₂polypeptide (positions 26-109), a pep27 polypeptide (positions 27-136),a F₁ polypeptide (positions 137-513), a trimerization domain (a Foldondomain) and a thrombin cleavage site (LVPRGS) and purification tags(his-tag (HHHHHH) and Strep Tag II (SAWSHPQFEK)). Thus, in additionalembodiments, the PreF antigen includes a recombinant RSV F proteinincluding a F₁ polypeptide and a F₂ polypeptide as set forth in any oneof the SEQ ID NOs listed in of row 1, 2, 3, 4, 5, 6, 7 or 8 of column 4of Table 6, wherein the PreF antigen is specifically bound by aprefusion specific antibody (e.g., D25 or AM22 antibody), and/orincludes a RSV F prefusion specific conformation (such as antigenic siteØ). In further embodiments, the PreF antigen includes a recombinant RSVF protein including a F₁ polypeptide and a F₂ polypeptide as set forthas positions 26-109 and 137-513, respectively, as set forth in any oneof the SEQ ID NOs listed in of row 1, 2, 3, 4, 5, 6, 7, or 8 of column 4of Table 6, wherein the PreF antigen is specifically bound by aprefusion specific antibody (e.g., D25 or AM22 antibody), and/orincludes a RSV F prefusion specific conformation (such as antigenic siteØ).

iii. Repacking Substitutions and N-Linked Glycosylation Substitutions

Additional embodiments concerning repacking amino acid substitutions andN-linked glycosylation amino acid substitutions are disclosed insections (II.B.1.a.iii) and (II.B.1.a.iv) on pages 53-58 and Tables 7and 8 of priority U.S. Provisional application No. 61/798,389, filedMar. 15, 2013, which is specifically incorporated by reference herein inits entirety.

b. Additional Modifications

In several embodiments, the PreF antigen includes a membrane anchoredform of the recombinant RSV F protein (e.g., with a transmembranedomain). In other embodiments, the PreF antigen includes a soluble formof the recombinant RSV F protein (e.g., without a transmembrane domainor other membrane anchor). It will be understood that there are severaldifferent approaches for generating a soluble or membrane anchoredrecombinant RSV F protein, including those discussed below. Examplesinclude introduction of a trimerization domain, introduction of cysteinepairs that can form a disulfide bond that stabilizes the C-terminalregion of F₁, and introduction of a transmembrane domain (e.g., forapplications including a membrane-anchored PreF antigen).

Further, as disclosed herein, the structure of the RSV F protein incomplex with D25 Fab (i.e., in a prefusion conformation) compared to thestructure of the postfusion RSV F protein (disclosed, e.g., in McLellanet al., J. Virol., 85, 7788, 2011, with coordinates deposited as PDBAccession No. 3RRR) show structural rearrangements between pre- andpost-fusion conformations in both the membrane-proximal andmembrane-distal lobes. Several embodiments include a modificationtargeted for stabilization of the membrane proximal lobe of the RSV Fprotein prefusion conformation. It will be understood that thesemodifications are not strictly necessary to stabilize a recombinant RSVF protein in a prefusion conformation, but that, in some instances, theyare combined with other prefusion stabilizing modifications, such asthose described above.

i. Trimerization Domain

In several embodiments, the PreF antigen includes a recombinant RSV Fprotein including an F1 polypeptide with a trimerization domain linkedto its C-terminus. In several embodiments, the trimerization domainpromotes trimerization of the three F1/F2 protomers in the recombinantRSV F protein. Several exogenous multimerization domains promote stabletrimers of soluble recombinant proteins: the GCN4 leucine zipper(Harbury et al. 1993 Science 262:1401-1407), the trimerization motiffrom the lung surfactant protein (Hoppe et al. 1994 FEBS Lett344:191-195), collagen (McAlinden et al. 2003 J Biol Chem278:42200-42207), and the phage T4 fibritin Foldon (Miroshnikov et al.1998 Protein Eng 11:329-414), any of which can be linked to the F1polypeptide in the PreF antigen to promote trimerization of therecombinant F protein, as long as the PreF antigen is specifically boundby a prefusion specific antibody (e.g., D25 or AM22 antibody), and/orincludes a RSV F prefusion specific conformation (such as antigenic siteØ).

In some examples, the PreF antigen includes a recombinant RSV F proteinincluding an F₁ polypeptide with a Foldon domain linked to itsC-terminus. In specific examples, the Foldon domain is a T4 fibritinFoldon domain such as the amino acid sequence GYIPEAPRDGQAYVRKDGEWVLLSTF(SEQ ID NO: 351), which adopts a β-propeller conformation, and can foldand trimerize in an autonomous way (Tao et al. 1997 Structure5:789-798). In some specific examples, a PreF antigen including arecombinant RSV F protein including a T4 fibritin Foldon domain,includes a F₂ polypeptide and an F₁ polypeptide linked to a Foldondomain as set forth in one of SEQ ID NOs: 185, or 189-303. Typically,the heterologous multimerization motif is positioned C-terminal to theF₁ domain. Optionally, the multimerization domain is connected to the F₁polypeptide via a linker, such as an amino acid linker, such as thesequence GG. The linker can also be a longer linker (for example,including the sequence GG, such as the amino acid sequence: GGSGGSGGS;SEQ ID NO: 352). Numerous conformationally neutral linkers are known inthe art that can be used in this context without disrupting theconformation of the PreF antigen.

In some embodiments, the PreF antigen includes a recombinant RSV Fprotein including any of the trimerization domain modifications listedabove combined with any of the modifications listed in section II.B.1.For example, in some embodiments, the PreF antigen includes arecombinant RSV F protein including any of the trimerization domainmodifications listed above in combination with one or more of thedisulfide bond modification listed in one of rows 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, or 51 of Table 5, and/or one or more of thecavity filling modifications listed in one of rows 1, 2, 3, 4, 5, 6, 7,or 8 of Table 6, and/or one or more of the repacking modificationslisted in one of rows 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, or 47 of Table 7,and/or one or more of the glycosylation modifications listed in one orrows 1, 2, 3, 4, 5, 6, 7, 8, or 9 of Table 8, wherein the PreF antigenis specifically bound by a prefusion specific antibody (e.g., D25 orAM22 antibody), and/or includes a RSV F prefusion specific conformation(such as antigenic site Ø).

For example, in some embodiments, the PreF antigen includes arecombinant RSV F protein stabilized in a RSV F protein prefusionconformation, and includes one or more disulfide bonds and a Foldondomain, wherein the F₂ polypeptide and the F₁ polypeptide linked to theFoldon domain include the amino acid sequence set forth as positions26-109 and 137-544, respectively, of any one of SEQ ID NO: 185, SEQ IDNO: 189, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 205, SEQ ID NO: 207,SEQ ID NO: 209, SEQ ID NO: 213, SEQ ID NO: 244, SEQ ID NO: 245, SEQ IDNO: 247, SEQ ID NO: 257, SEQ ID NO: 264, SEQ ID NO: 265, SEQ ID NO: 266,SEQ ID NO: 267, SEQ ID NO: 268, SEQ ID NO: 269, SEQ ID NO: 270, SEQ IDNO: 271, SEQ ID NO: 272, SEQ ID NO: 273, SEQ ID NO: 274, SEQ ID NO: 275,SEQ ID NO: 277, SEQ ID NO: 278, SEQ ID NO: 279, SEQ ID NO: 280, SEQ IDNO: 281, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 302, SEQ ID NO: 303,SEQ ID NO: 190, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 243, SEQ IDNO: 246, SEQ ID NO: 276, SEQ ID NO: 283, SEQ ID NO: 285, or SEQ ID NO:263; or positions 26-109 and 137-545, respectively, of any one of SEQ IDNO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 262,SEQ ID NO: 296, SEQ ID NO: 297, SEQ ID NO: 298, or SEQ ID NO: 299,wherein the PreF antigen is specifically bound by a prefusion specificantibody (e.g., D25 or AM22 antibody), and/or includes a RSV F prefusionspecific conformation (such as antigenic site Ø).

In additional embodiments, the PreF antigen includes a recombinant RSV Fprotein stabilized in a RSV F protein prefusion conformation, andincludes one or more cavity-filling amino acid substitution and a Foldondomain, wherein the F₂ polypeptide and the F₁ polypeptide linked to theFoldon domain include the amino acid sequence set forth as positions26-109 and 137-544, respectively, of any one of SEQ ID NO: 191, SEQ IDNO: 193, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 248, SEQ ID NO: 192,SEQ ID NO: 195, or SEQ ID NO: 194; wherein the PreF antigen isspecifically bound by a prefusion specific antibody (e.g., D25 or AM22antibody), and/or includes a RSV F prefusion specific conformation (suchas antigenic site Ø).

In additional embodiments, the PreF antigen includes a recombinant RSV Fprotein stabilized in a RSV F protein prefusion conformation, andincludes one or more repacking amino acid substitutions and a Foldondomain, wherein the F₂ polypeptide and the F₁ polypeptide linked to theFoldon domain include the amino acid sequence set forth as positions26-109 and 137-544, respectively, of any one of SEQ ID NO: 249, SEQ IDNO: 250, SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID NO: 253, SEQ ID NO: 254,SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 288, SEQ ID NO: 289, SEQ IDNO: 290, SEQ ID NO: 291, SEQ ID NO: 292, SEQ ID NO: 293, SEQ ID NO: 294,SEQ ID NO: 295, SEQ ID NO: 296, SEQ ID NO: 297, SEQ ID NO: 326, SEQ IDNO: 327, SEQ ID NO: 328, SEQ ID NO: 329, SEQ ID NO: 330, SEQ ID NO: 331,SEQ ID NO: 332, SEQ ID NO: 333, SEQ ID NO: 334, SEQ ID NO: 335, SEQ IDNO: 336, or SEQ ID NO: 337; wherein the PreF antigen is specificallybound by a prefusion specific antibody (e.g., D25 or AM22 antibody),and/or includes a RSV F prefusion specific conformation (such asantigenic site Ø).

In further embodiments, the PreF antigen includes a recombinant RSV Fprotein stabilized in a RSV F protein prefusion conformation, andincludes one or more N-linked glycosylation sites and a Foldon domain,wherein the F₂ polypeptide and the F₁ polypeptide linked to the Foldondomain include the amino acid sequence set forth as positions 26-109 and137-544, respectively, of any one of SEQ ID NOs selected from the groupconsisting of SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO:203, SEQ ID NO: 204, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, orSEQ ID NO: 217; wherein the PreF antigen is specifically bound by aprefusion specific antibody (e.g., D25 or AM22 antibody), and/orincludes a RSV F prefusion specific conformation (such as antigenic siteØ).

ii. Disulfide Bonds

In some embodiments, the PreF antigen includes a recombinant RSV Fprotein including a F1 polypeptide including one or more disulfide bondsthat are used to stabilize the membrane proximal lobe of the recombinantRSV F protein. The cysteine residues that form the disulfide bond can beintroduced into the recombinant RSV F protein by one or more amino acidsubstitutions. For example, in some embodiments, a single amino acidsubstitution introduces a cysteine that forms a disulfide bond with acysteine residue present in the native RSV F protein sequence. Inadditional embodiments, two cysteine residues are introduced into anative RSV sequence to form the disulfide bond. The location of thecysteine (or cysteines) of a disulfide bond to stabilize the membraneproximal lobe of the RSV F protein in a prefusion conformation canreadily be determined by the person of ordinary skill in the art usingmethods described herein and familiar to the skilled artisan.

In some embodiments, the PreF antigen includes a recombinant RSV Fprotein including a disulfide bond between cysteine residues located atRSV F positions 486 and 487, or between cysteine residues located at RSVF positions 512 and 513, wherein the PreF antigen is specifically boundby a prefusion specific antibody (e.g., D25 or AM22 antibody), and/orincludes a RSV F prefusion specific conformation (such as antigenic siteØ). In some such embodiments, the F₁ polypeptide includes D486C andE487C substitutions, or L512C and L513C substitutions, respectively.

In some embodiments, amino acids can be inserted (or deleted) from the Fprotein sequence to adjust the alignment of residues in the F proteinstructure, such that particular residue pairs are within a sufficientlyclose distance to form an disulfide bond. In some such embodiments, thePreF antigen includes a recombinant RSV F protein including a disulfidebond between cysteine residues located at 486 and 487; with a prolineinsertion between positions 486 and 487, wherein the PreF antigen isspecifically bound by a prefusion specific antibody (e.g., D25 or AM22antibody), and/or includes a RSV F prefusion specific conformation (suchas antigenic site Ø). In some such embodiments, the F₁ polypeptideincludes D486C and E487C substitutions, and a proline insertion betweenpositions 486 and 487.

In additional embodiments, the PreF antigen includes a recombinant RSV Fprotein including a disulfide bond between a cysteine residue located atposition 493 and a cysteine residue inserted between positions 329 and330, wherein the PreF antigen is specifically bound by a prefusionspecific antibody (e.g., D25 or AM22 antibody), and/or includes a RSV Fprefusion specific conformation (such as antigenic site Ø). In some suchembodiments, the F₁ polypeptide includes S493C substitution, and acysteine residue inserted between positions 329 and 330.

In additional embodiments, the PreF antigen includes a recombinant RSV Fprotein including a disulfide bond between a cysteine residue located atposition 493 and a cysteine residue inserted between positions 329 and330, and further includes a glycine insertion between residues 492 and493, wherein the PreF antigen is specifically bound by a prefusionspecific antibody (e.g., D25 or AM22 antibody), and/or includes a RSV Fprefusion specific conformation (such as antigenic site Ø). In some suchembodiments, the F₁ polypeptide includes S493C substitution, a cysteineresidue inserted between positions 329 and 330, and a glycine insertionbetween residues 492 and 493

In some embodiments, the PreF antigen includes a recombinant RSV Fprotein including any of the above disulfide bond modifications forstabilizing the membrane proximal lobe of the RSV F protein, combinedwith any of the stabilization modifications listed in section II.B.1. Insome embodiments, the PreF antigen includes a recombinant RSV F proteinincluding any of the disulfide bond modifications for stabilizing themembrane proximal lobe of the RSV F protein listed above in combinationwith one or more of the disulfide bond modifications listed in one ofrows 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 51 of Table 5,and/or one or more of the cavity filling modifications listed in one ofrows 1, 2, 3, 4, 5, 6, 7, or 8 of Table 6, and/or one or more of therepacking modifications listed in one of rows 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, or 47 of Table 7, and/or one or more of the glycosylationmodifications listed in one or rows 1, 2, 3, 4, 5, 6, 7, 8, or 9 ofTable 8, wherein the PreF antigen is specifically bound by a prefusionspecific antibody (e.g., D25 or AM22 antibody), and/or includes a RSV Fprefusion specific conformation (such as antigenic site Ø).

iii. Transmembrane Domains

In some embodiments, the recombinant RSV F protein includes atransmembrane domain linked to the F₁ polypeptide, for example, for anapplication including a membrane anchored PreF antigen. For example, thepresence of the transmembrane sequences is useful for expression as atransmembrane protein for membrane vesicle preparation. Thetransmembrane domain can be linked to a F₁ protein containing any of thestabilizing mutations provided herein, for example, those describedabove, such as a F₁ protein with a S155C/S290C cysteine substitution.Additionally, the transmembrane domain can be further linked to a RSV F₁cytosolic tail. Examples include a signal peptide, F₂ polypeptide(positions 26-109), pep27 polypeptide (positions 27-136), F₁ polypeptide(positions 137-513), a RSV transmembrane domain are provided as SEQ IDNO: 323 (without a cytosolic domain) and SEQ ID NO: 324 (with acytosolic domain).

In some embodiments, the PreF antigen includes a recombinant RSV Fprotein including an F1 polypeptide linked to a transmembrane domain,combined with any of the stabilization modifications listed in sectionII.B.1. For example, in some embodiments, the PreF antigen includes arecombinant RSV F protein including an F₁ polypeptide linked to atransmembrane domain, and further includes one or more of the disulfidebond modification listed in one of rows 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, or 51 of Table 5, and/or one or more of the cavityfilling modifications listed in one of rows 1, 2, 3, 4, 5, 6, 7, or 8 ofTable 6, and/or one or more of the repacking modifications listed in oneof rows 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, or 47 of Table 7, and/or one ormore of the glycosylation modifications listed in one or rows 1, 2, 3,4, 5, 6, 7, 8, or 9 of Table 8, wherein the PreF antigen is specificallybound by a prefusion specific antibody (e.g., D25 or AM22 antibody),and/or includes a RSV F prefusion specific conformation (such asantigenic site Ø).

iv. Antigenic Sites

In some embodiments, the PreF antigen includes a recombinant RSV Fprotein that is stabilized in a prefusion conformation and includesfurther modification to eliminate a known antigenic site other thanantigenic site Ø. For example, the recombinant RSV F protein can includea modification that disrupts antigenic site I, II or IV. Suchmodifications can be identified, for example, by binding of antibodiesspecific for these sites.

2. Epitope-Scaffold Proteins

In several embodiments, the PreF antigen includes an epitope-scaffoldprotein including a RSV F protein prefusion specific epitope in aprefusion specific conformation. In some examples, the epitope scaffoldprotein includes any of the recombinant RSV F proteins stabilized in aprefusion conformation as disclosed herein. The prefusion specificepitope can be placed anywhere in the scaffold protein (for example, onthe N-terminus, C-terminus, or an internal loop), as long as the PreFantigen including the epitope scaffold protein is specifically bound bya prefusion specific antibody (e.g., D25 or AM22 antibody), and/orincludes a RSV F prefusion specific conformation (such as antigenic siteØ).

Methods for identifying and selecting scaffolds are disclosed herein andknown to the person of ordinary skill in the art. For example, methodsfor superposition, grafting and de novo design of epitope-scaffolds aredisclosed in U.S. Patent Application Publication No. 2010/0068217,incorporated by reference herein in its entirety.

“Superposition” epitope-scaffolds are based on scaffold proteins havingan exposed segment with similar conformation as the target epitope—thebackbone atoms in this “superposition-region” can be structurallysuperposed onto the target epitope with minimal root mean squaredeviation (RMSD) of their coordinates. Suitable scaffolds are identifiedby computationally searching through a library of protein crystalstructures; epitope-scaffolds are designed by putting the epitoperesidues in the superposition region and making additional mutations onthe surrounding surface of the scaffold to prevent clash or otherinteractions with the antibody.

“Grafting” epitope-scaffolds utilize scaffold proteins that canaccommodate replacement of an exposed segment with the crystallizedconformation of the target epitope. For each suitable scaffoldidentified by computationally searching through all protein crystalstructures, an exposed segment is replaced by the target epitope and thesurrounding sidechains are redesigned (mutated) to accommodate andstabilize the inserted epitope. Finally, as with superpositionepitope-scaffolds, mutations are made on the surface of the scaffold andoutside the epitope, to prevent clash or other interactions with theantibody. Grafting scaffolds require that the replaced segment andinserted epitope have similar translation and rotation transformationsbetween their N- and C-termini, and that the surrounding peptidebackbone does not clash with the inserted epitope. One differencebetween grafting and superposition is that grafting attempts to mimicthe epitope conformation exactly, whereas superposition allows for smallstructural deviations.

“De novo” epitope-scaffolds are computationally designed from scratch tooptimally present the crystallized conformation of the epitope. Thismethod is based on computational design of a novel fold (Kuhlman, B. etal. 2003 Science 302:1364-1368). The de novo allows design of immunogensthat are both minimal in size, so they do not present unwanted epitopes,and also highly stable against thermal or chemical denaturation.

The scaffold can be a heterologous scaffold. In several embodiments, thenative scaffold protein (without epitope insertion) is not a viralenvelope protein. In additional embodiments, the scaffold protein is nota RSV protein. In still further embodiments, the scaffold protein is nota viral protein.

In additional embodiments, the epitope-scaffold protein includes theamino acid sequence set forth as any one of SEQ ID NOs: 341-343, or apolypeptide with at least 80% sequence identity (such as at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98% orat least 99% sequence identity) to any one of SEQ ID NOs: 341-343, andwherein the epitope-scaffold protein is specifically bound by aprefusion specific antibody (e.g., D25 or AM22 antibody), and/orincludes a RSV F prefusion specific conformation (such as antigenic siteØ). In additional embodiments, the RSV F protein is any one of SEQ IDNOs: 341-343, wherein the amino acid sequence of the RSV F protein hasup to 20 amino acid substitutions, and wherein the epitope scaffoldprotein is specifically bound by a prefusion specific antibody (e.g.,D25 or AM22 antibody), and/or includes a RSV F prefusion specificconformation (such as antigenic site Ø), in the absence of binding bythe corresponding prefusion specific antibody (e.g., D25 or AM22antibody). Alternatively, the polypeptide can have none, or up to 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 amino acidsubstitutions.

The recombinant RSV F protein stabilized in a prefusion conformation canbe placed anywhere in the scaffold, as long as the resultingepitope-scaffold protein is specifically bound by a prefusion specificantibody (e.g., D25 or AM22 antibody), and/or includes a RSV F prefusionspecific conformation (such as antigenic site Ø), in the absence ofbinding by the corresponding prefusion specific antibody (e.g., D25 orAM22 antibody). Methods for determining if a particular epitope-scaffoldprotein is specifically bound by a prefusion specific antibody (e.g.,D25 or AM22 antibody) are disclosed herein and known to the person ofordinary skill in the art (see, for example, International ApplicationPub. Nos. WO 2006/091455 and WO 2005/111621). In addition, the formationof an antibody-antigen complex can be assayed using a number ofwell-defined diagnostic assays including conventional immunoassayformats to detect and/or quantitate antigen-specific antibodies. Suchassays include, for example, enzyme immunoassays, e.g., ELISA,cell-based assays, flow cytometry, radioimmunoassays, andimmunohistochemical staining. Numerous competitive and non-competitiveprotein binding assays are known in the art and many are commerciallyavailable. Methods for determining if a particular epitope-scaffoldprotein includes a RSV F prefusion specific conformation (such asantigenic site Ø), in the absence of binding by the correspondingprefusion specific antibody (e.g., D25 or AM22 antibody) are alsodescribed herein and further known to the person of ordinary skill inthe art.

3. Virus-Like Particles

In some embodiments, a virus-like particle (VLP) is provided thatincludes a disclosed recombinant RSV F protein stabilized in a prefusionconformation. VLPs lack the viral components that are required for virusreplication and thus represent a highly attenuated form of a virus. TheVLP can display a polypeptide (e.g., a recombinant RSV F proteinstabilized in a prefusion conformation) that is capable of eliciting animmune response to RSV when administered to a subject. Virus likeparticles and methods of their production are known and familiar to theperson of ordinary skill in the art, and viral proteins from severalviruses are known to form VLPs, including human papillomavirus, HIV(Kang et al., Biol. Chem. 380: 353-64 (1999)), Semliki-Forest virus(Notka et al., Biol. Chem. 380: 341-52 (1999)), human polyomavirus(Goldmann et al., J. Virol. 73: 4465-9 (1999)), rotavirus (Jiang et al.,Vaccine 17: 1005-13 (1999)), parvovirus (Casal, Biotechnology andApplied Biochemistry, Vol 29, Part 2, pp 141-150 (1999)), canineparvovirus (Hurtado et al., J. Virol. 70: 5422-9 (1996)), hepatitis Evirus (Li et al., J. Virol. 71: 7207-13 (1997)), and Newcastle diseasevirus. For example, a chimeric VLP containing a RSV antigen and can be aNewcastle disease virus-based VLP. Newcastle disease based VLPs havepreviously been shown to elicit a neutralizing immune response to RSV inmice. The formation of such VLPs can be detected by any suitabletechnique. Examples of suitable techniques known in the art fordetection of VLPs in a medium include, e.g., electron microscopytechniques, dynamic light scattering (DLS), selective chromatographicseparation (e.g., ion exchange, hydrophobic interaction, and/or sizeexclusion chromatographic separation of the VLPs) and density gradientcentrifugation.

4. Protein Nanoparticles

In some embodiments a protein nanoparticle is provided that includes oneor more of any of the disclosed recombinant RSV F protein stabilized ina prefusion conformation, wherein the protein nanoparticle isspecifically bound by a prefusion specific antibody (e.g., D25 or AM22antibody), and/or includes a RSV F prefusion specific conformation (suchas antigenic site Ø). Non-limiting example of nanoparticles includeferritin nanoparticles, an encapsulin nanoparticles and Sulfur OxygenaseReductase (SOR) nanoparticles, which are comprised of an assembly ofmonomeric subunits including ferritin proteins, encapsulin proteins andSOR proteins, respectively. To construct protein nanoparticles includingthe disclosed recombinant RSV F protein stabilized in a prefusionconformation, the antigen is linked to a subunit of the proteinnanoparticle (such as a ferritin protein, an encapsulin protein or a SORprotein). The fusion protein self-assembles into a nanoparticle underappropriate conditions.

In some embodiments, any of the disclosed recombinant RSV F proteinsstabilized in a prefusion conformation are linked to a ferritinpolypeptide or hybrid of different ferritin polypeptides to construct aferritin protein nanoparticle, wherein the ferritin nanoparticle isspecifically bound by a prefusion specific antibody (e.g., D25 or AM22antibody), and/or includes a RSV F prefusion specific conformation (suchas antigenic site Ø). Ferritin is a globular protein that is found inall animals, bacteria, and plants, and which acts primarily to controlthe rate and location of polynuclear Fe(III)₂O₃ formation through thetransportation of hydrated iron ions and protons to and from amineralized core. The globular form of ferritin is made up of monomericsubunits, which are polypeptides having a molecule weight ofapproximately 17-20 kDa. An example of the sequence of one suchmonomeric subunit is represented by SEQ ID NO: 353. Each monomericsubunit has the topology of a helix bundle which includes a fourantiparallel helix motif, with a fifth shorter helix (the c-terminalhelix) lying roughly perpendicular to the long axis of the 4 helixbundle. According to convention, the helices are labeled ‘A, B, C, D &E’ from the N-terminus respectively. The N-terminal sequence liesadjacent to the capsid three-fold axis and extends to the surface, whilethe E helices pack together at the four-fold axis with the C-terminusextending into the capsid core. The consequence of this packing createstwo pores on the capsid surface. It is expected that one or both ofthese pores represent the point by which the hydrated iron diffuses intoand out of the capsid. Following production, these monomeric subunitproteins self-assemble into the globular ferritin protein. Thus, theglobular form of ferritin comprises 24 monomeric, subunit proteins, andhas a capsid-like structure having 432 symmetry. Methods of constructingferritin nanoparticles are known to the person of ordinary skill in theart and are further described herein (see, e.g., Zhang, Int. J. Mol.Sci., 12:5406-5421, 2011, which is incorporated herein by reference inits entirety).

In specific examples, the ferritin polypeptide is E. coli ferritin,Helicobacter pylori ferritin, human light chain ferritin, bullfrogferritin or a hybrid thereof, such as E. coli-human hybrid ferritin, E.coli-bullfrog hybrid ferritin, or human-bullfrog hybrid ferritin.Exemplary amino acid sequences of ferritin polypeptides and nucleic acidsequences encoding ferritin polypeptides for use in the disclosed RSV Fprotein antigens stabilized in a prefusion conformation can be found inGENBANK®, for example at accession numbers ZP_(—)03085328,ZP_(—)06990637, EJB64322.1, AAA35832, NP_(—)000137 AAA49532, AAA49525,AAA49524 and AAA49523, which are specifically incorporated by referenceherein in their entirety as available Feb. 28, 2013. In one embodiment,any of the disclosed recombinant RSV F proteins stabilized in aprefusion conformation is linked to a ferritin protein including anamino acid sequence at least 80% (such as at least 85%, at least 90%, atleast 95%, or at least 97%) identical to amino acid sequence set forthas SEQ ID NO: 353. A specific example of the disclosed recombinant RSV Fproteins stabilized in a prefusion conformation linked to a ferritinprotein include the amino acid sequence set forth as SEQ ID NO: 350.

In additional embodiments, any of the disclosed RSV F protein antigensstabilized in a prefusion conformation are linked to an encapsulinpolypeptide to construct an encapsulin nanoparticle, wherein theencapsulin nanoparticle is specifically bound by a prefusion specificantibody (e.g., D25 or AM22 antibody), and/or includes a RSV F prefusionspecific conformation (such as antigenic site Ø). Encapsulin proteinsare a conserved family of bacterial proteins also known as linocin-likeproteins that form large protein assemblies that function as a minimalcompartment to package enzymes. The encapsulin assembly is made up ofmonomeric subunits, which are polypeptides having a molecule weight ofapproximately 30 kDa. An example of the sequence of one such monomericsubunit is provided as SEQ ID NO: 354. Following production, themonomeric subunits self-assemble into the globular encapsulin assemblyincluding 60 monomeric subunits. Methods of constructing encapsulinnanoparticles are known to the person of ordinary skill in the art, andfurther described herein (see, for example, Sutter et al., NatureStruct. and Mol. Biol., 15:939-947, 2008, which is incorporated byreference herein in its entirety). In specific examples, the encapsulinpolypeptide is bacterial encapsulin, such as E. coli or Thermotogamaritime encapsulin. An exemplary encapsulin sequence for use with thedisclosed RSV F protein antigens stabilized in a prefusion conformationis set forth as SEQ ID NO: 354.

In additional embodiments, any of the disclosed recombinant RSV Fproteins stabilized in a prefusion conformation are linked to a SulfurOxygenase Reductase (SOR) polypeptide to construct a SOR nanoparticle,wherein the SOR nanoparticle is specifically bound by a prefusionspecific antibody (e.g., D25 or AM22 antibody), and/or includes a RSV Fprefusion specific conformation (such as antigenic site Ø). SOR proteinsare microbial proteins (for example from the thermoacidophilic archaeonAcidianus ambivalens that form 24 subunit protein assemblies. Methods ofconstructing SOR nanoparticles are known to the person of ordinary skillin the art (see, e.g., Urich et al., Science, 311:996-1000, 2006, whichis incorporated by reference herein in its entirety). Specific examplesof the disclosed recombinant RSV F proteins stabilized in a prefusionconformation linked to a SOR protein include the amino acid sequencesset forth as SEQ ID NO: 344 and SEQ ID NO: 345.

In additional embodiments, any of the disclosed recombinant RSV Fproteins stabilized in a prefusion conformation are linked to a Lumazinesynthase polypeptide to construct a Lumazine synthase nanoparticle,wherein the Lumazine synthase nanoparticle is specifically bound by aprefusion specific antibody (e.g., D25 or AM22 antibody), and/orincludes a RSV F prefusion specific conformation (such as antigenic siteØ). Specific examples of the disclosed recombinant RSV F proteinsstabilized in a prefusion conformation linked to a Lumazine synthaseprotein include the amino acid sequences set forth as SEQ ID NOs:346-348.

In additional embodiments, any of the disclosed recombinant RSV Fproteins stabilized in a prefusion conformation are linked to a pyruvatedehydrogenase polypeptide to construct a pyruvate dehydrogenasenanoparticle, wherein the pyruvate dehydrogenase nanoparticle isspecifically bound by a prefusion specific antibody (e.g., D25 or AM22antibody), and/or includes a RSV F prefusion specific conformation (suchas antigenic site Ø). A specific example of the disclosed recombinantRSV F proteins stabilized in a prefusion conformation linked to apyruvate dehydrogenase protein include the amino acid sequence set forthas SEQ ID NO: 349.

In some examples, any of the disclosed recombinant RSV F proteinsstabilized in a prefusion conformation is linked to the N- or C-terminusof a ferritin, encapsulin, SOR, lumazine synthase or pyruvatedehydrogenase protein, for example with a linker, such as a Ser-Glylinker. When the constructs have been made in HEK 293 Freestyle cells,the fusion proteins are secreted from the cells and self-assembled intoparticles. The particles can be purified using known techniques, forexample by a few different chromatography procedures, e.g. Mono Q (anionexchange) followed by size exclusion (SUPEROSE® 6) chromatography.

Several embodiments include a monomeric subunit of a ferritin,encapsulin, SOR, lumazine synthase or pyruvate dehydrogenase protein, orany portion thereof which is capable of directing self-assembly ofmonomeric subunits into the globular form of the protein. Amino acidsequences from monomeric subunits of any known ferritin, encapsulin,SOR, lumazine synthase or pyruvate dehydrogenase protein can be used toproduce fusion proteins with the disclosed recombinant RSV F proteinsstabilized in a prefusion conformation, so long as the monomeric subunitis capable of self-assembling into a nanoparticle displaying therecombinant RSV F proteins stabilized in a prefusion conformation on itssurface.

The fusion proteins need not comprise the full-length sequence of amonomeric subunit polypeptide of a ferritin, encapsulin, SOR, lumazinesynthase or pyruvate dehydrogenase protein. Portions, or regions, of themonomeric subunit polypeptide can be utilized so long as the portioncomprises amino acid sequences that direct self-assembly of monomericsubunits into the globular form of the protein.

In some embodiments, it may be useful to engineer mutations into theamino acid sequence of the monomeric ferritin, encapsulin, SOR, lumazinesynthase or pyruvate dehydrogenase subunits. For example, it may beuseful to alter sites such as enzyme recognition sites or glycosylationsites in order to give the fusion protein beneficial properties (e.g.,half-life).

It will be understood by those skilled in the art that fusion of any ofthe disclosed recombinant RSV F proteins stabilized in a prefusionconformation to the ferritin, encapsulin, SOR, lumazine synthase orpyruvate dehydrogenase protein should be done such that the disclosedrecombinant RSV F proteins stabilized in a prefusion conformationportion of the fusion protein does not interfere with self-assembly ofthe monomeric ferritin, encapsulin, SOR, lumazine synthase or pyruvatedehydrogenase subunits into the globular protein, and that the ferritin,encapsulin, SOR, lumazine synthase or pyruvate dehydrogenase proteinportion of the fusion protein does not interfere with the ability of thedisclosed recombinant RSV F protein antigen stabilized in a prefusionconformation to elicit an immune response to RSV. In some embodiments,the ferritin, encapsulin, SOR, lumazine synthase or pyruvatedehydrogenase protein and disclosed recombinant RSV F protein stabilizedin a prefusion conformation can be joined together directly withoutaffecting the activity of either portion. In other embodiments, theferritin, encapsulin, SOR, lumazine synthase or pyruvate dehydrogenaseprotein and the recombinant RSV F protein stabilized in a prefusionconformation are joined using a linker (also referred to as a spacer)sequence. The linker sequence is designed to position the ferritin,encapsulin, SOR, lumazine synthase or pyruvate dehydrogenase portion ofthe fusion protein and the disclosed recombinant RSV F proteinstabilized in a prefusion conformation portion of the fusion protein,with regard to one another, such that the fusion protein maintains theability to assemble into nanoparticles, and also elicit an immuneresponse to RSV. In several embodiments, the linker sequences compriseamino acids. Preferable amino acids to use are those having small sidechains and/or those which are not charged. Such amino acids are lesslikely to interfere with proper folding and activity of the fusionprotein. Accordingly, preferred amino acids to use in linker sequences,either alone or in combination are serine, glycine and alanine Oneexample of such a linker sequence is SGG. Amino acids can be added orsubtracted as needed. Those skilled in the art are capable ofdetermining appropriate linker sequences for construction of proteinnanoparticles.

In certain embodiments, the protein nanoparticles have a molecularweight of from 100 to 5000 kDa, such as approximately 500 to 4600 kDa.In some embodiments, a Ferritin nanoparticle has an approximatemolecular weight of 650 kDa, an Encapsulin nanoparticle has anapproximate molecular weight of 2100 kDa, a SOR nanoparticle has anapproximate molecular weight of 1000 kDa, a lumazine synthase particlehas an approximate molecular weight of 4000 kDa, and a pyruvatedehydrogenase particle has an approximate molecular weight of 4600 kDa,when the protein nanoparticle include a recombinant RSV F proteinstabilized in a prefusion conformation.

The disclosed recombinant RSV F proteins stabilized in a prefusionconformation linked to ferritin, encapsulin, SOR, lumazine synthase orpyruvate dehydrogenase proteins can self-assemble into multi-subunitprotein nanoparticles, termed ferritin nanoparticles, encapsulinnanoparticles, SOR nanoparticles, lumazine synthase nanoparticles, andpyruvate dehydrogenase nanoparticles, respectively. The nanoparticlesinclude the disclosed recombinant RSV F proteins stabilized in aprefusion conformation have substantially the same structuralcharacteristics as the native ferritin, encapsulin, SOR, lumazinesynthase or pyruvate dehydrogenase nanoparticles that do not include thedisclosed recombinant RSV F proteins stabilized in a prefusionconformation. That is, they contain 24, 60, 24, 60, or 60 subunits(respectively) and have similar corresponding symmetry. In the case ofnanoparticles constructed of monomer subunits including a disclosedrecombinant RSV F protein stabilized in a prefusion conformation, suchnanoparticles are specifically bound by a prefusion specific antibody(e.g., D25 or AM22 antibody), and/or includes a RSV F prefusion specificconformation (such as antigenic site Ø).

C. Polynucleotides Encoding Antigens

Polynucleotides encoding the disclosed PreF antigens (e.g., arecombinant RSV F protein stabilized in a prefusion conformation, orepitope-scaffold protein, or virus-like particle or protein nanoparticlecontaining such proteins) are also provided. These polynucleotidesinclude DNA, cDNA and RNA sequences which encode the antigen.

In some embodiments, the nucleic acid molecule encodes a precursor F₀polypeptide that, when expressed in an appropriate cell, is processedinto a disclosed PreF antigen. In some embodiments, the nucleic acidmolecule encodes a precursor F₀ polypeptide that, when expressed in anappropriate cell, is processed into a disclosed PreF antigen, whereinthe precursor F₀ polypeptide includes, from N- to C-terminus, a signalpeptide, a F₂ polypeptide, a Pep27 polypeptide, and a F₁ polypeptide. Insome embodiments, the Pep27 polypeptide includes the amino acid sequenceset forth as positions 110-136 of any one SEQ ID NOs: 1-184, wherein theamino acid positions correspond to the amino acid sequence of areference F₀ polypeptide set forth as SEQ ID NO: 124. In someembodiments, the signal peptide includes the amino acid sequence setforth as positions 1-25 of any one SEQ ID NOs: 1-184, wherein the aminoacid positions correspond to the amino acid sequence of a reference F₀polypeptide set forth as SEQ ID NO: 124.

In some embodiments, the nucleic acid molecule encodes a precursor F₀polypeptide that, when expressed in an appropriate cell, is processedinto a disclosed PreF antigen, wherein the precursor F₀ polypeptideincludes the amino acid sequence set forth as any one of SEQ ID NOs:185, or 189-303.

Methods for the manipulation and insertion of the nucleic acids of thisdisclosure into vectors are well known in the art (see for example,Sambrook et al., Molecular Cloning, a Laboratory Manual, 2d edition,Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989, and Ausubel etal., Current Protocols in Molecular Biology, Greene PublishingAssociates and John Wiley & Sons, New York, N.Y., 1994).

A nucleic acid encoding PreF antigens (e.g., a recombinant RSV F proteinstabilized in a prefusion conformation, or epitope-scaffold protein, orvirus-like particle or protein nanoparticle containing such proteins)can be cloned or amplified by in vitro methods, such as the polymerasechain reaction (PCR), the ligase chain reaction (LCR), thetranscription-based amplification system (TAS), the self-sustainedsequence replication system (3SR) and the Qβ replicase amplificationsystem (QB). For example, a polynucleotide encoding the protein can beisolated by polymerase chain reaction of cDNA using primers based on theDNA sequence of the molecule. A wide variety of cloning and in vitroamplification methodologies are well known to persons skilled in theart. PCR methods are described in, for example, U.S. Pat. No. 4,683,195;Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 51:263, 1987; andErlich, ed., PCR Technology, (Stockton Press, NY, 1989). Polynucleotidesalso can be isolated by screening genomic or cDNA libraries with probesselected from the sequences of the desired polynucleotide understringent hybridization conditions.

The polynucleotides encoding PreF antigens (e.g., a recombinant RSV Fprotein stabilized in a prefusion conformation, or epitope-scaffoldprotein, or virus-like particle or protein nanoparticle containing suchproteins) include a recombinant DNA which is incorporated into a vectorinto an autonomously replicating plasmid or virus or into the genomicDNA of a prokaryote or eukaryote, or which exists as a separate molecule(such as a cDNA) independent of other sequences. The nucleotides can beribonucleotides, deoxyribonucleotides, or modified forms of eithernucleotide. The term includes single and double forms of DNA.

DNA sequences encoding PreF antigens (e.g., a recombinant RSV F proteinstabilized in a prefusion conformation, or epitope-scaffold protein, orvirus-like particle or protein nanoparticle containing such proteins)can be expressed in vitro by DNA transfer into a suitable host cell. Thecell may be prokaryotic or eukaryotic. The term also includes anyprogeny of the subject host cell. It is understood that all progeny maynot be identical to the parental cell since there may be mutations thatoccur during replication. Methods of stable transfer, meaning that theforeign DNA is continuously maintained in the host, are known in theart.

Polynucleotide sequences encoding PreF antigens (e.g., a recombinant RSVF protein stabilized in a prefusion conformation, or epitope-scaffoldprotein, or virus-like particle or protein nanoparticle containing suchproteins) can be operatively linked to expression control sequences. Anexpression control sequence operatively linked to a coding sequence isligated such that expression of the coding sequence is achieved underconditions compatible with the expression control sequences. Theexpression control sequences include, but are not limited to,appropriate promoters, enhancers, transcription terminators, a startcodon (i.e., ATG) in front of a protein-encoding gene, splicing signalfor introns, maintenance of the correct reading frame of that gene topermit proper translation of mRNA, and stop codons.

Hosts can include microbial, yeast, insect and mammalian organisms.Methods of expressing DNA sequences having eukaryotic or viral sequencesin prokaryotes are well known in the art. Non-limiting examples ofsuitable host cells include bacteria, archea, insect, fungi (forexample, yeast), plant, and animal cells (for example, mammalian cells,such as human). Exemplary cells of use include Escherichia coli,Bacillus subtilis, Saccharomyces cerevisiae, Salmonella typhimurium, SF9cells, C129 cells, 293 cells, Neurospora, and immortalized mammalianmyeloid and lymphoid cell lines. Techniques for the propagation ofmammalian cells in culture are well-known (see, Jakoby and Pastan (eds),1979, Cell Culture. Methods in Enzymology, volume 58, Academic Press,Inc., Harcourt Brace Jovanovich, N.Y.). Examples of commonly usedmammalian host cell lines are VERO and HeLa cells, CHO cells, and WI38,BHK, and COS cell lines, although cell lines may be used, such as cellsdesigned to provide higher expression, desirable glycosylation patterns,or other features. In some embodiments, the host cells include HEK293cells or derivatives thereof, such as GnTI^(−/−) cells (ATCC® No.CRL-3022).

Transformation of a host cell with recombinant DNA can be carried out byconventional techniques as are well known to those skilled in the art.Where the host is prokaryotic, such as, but not limited to, E. coli,competent cells which are capable of DNA uptake can be prepared fromcells harvested after exponential growth phase and subsequently treatedby the CaCl₂ method using procedures well known in the art.Alternatively, MgCl₂ or RbCl can be used. Transformation can also beperformed after forming a protoplast of the host cell if desired, or byelectroporation.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate coprecipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or viral vectors can be used. Eukaryotic cells can also beco-transformed with polynucleotide sequences encoding a disclosedantigen, and a second foreign DNA molecule encoding a selectablephenotype, such as the herpes simplex thymidine kinase gene. Anothermethod is to use a eukaryotic viral vector, such as simian virus 40(SV40) or bovine papilloma virus, to transiently infect or transformeukaryotic cells and express the protein (see for example, EukaryoticViral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).

D. Viral Vectors

The nucleic acid molecules encoding a recombinant RSV F proteinstabilized in a prefusion conformation can be included in a viralvector, for example for expression of the antigen in a host cell, or forimmunization of a subject as disclosed herein. In some embodiments, theviral vectors are administered to a subject as part of a prime-boostvaccination. In several embodiments, the viral vectors are included in avaccine, such as a primer vaccine or a booster vaccine for use in aprime-boost vaccination.

In several examples, the viral vector encoding the recombinant RSV Fprotein stabilized in a prefusion conformation can bereplication-competent. For example, the viral vector can have a mutation(e.g., insertion of nucleic acid encoding the PreF antigen) in the viralgenome that does not inhibit viral replication in host cells. The viralvector also can be conditionally replication-competent. In otherexamples, the viral vector is replication-deficient in host cells.

In several embodiments, the recombinant RSV F protein stabilized in aprefusion conformation is expressed by a viral vector that can bedelivered via the respiratory tract. For example, a paramyxovirus (PIV)vector, such as bovine parainfluenza virus (BPIV) vector (e.g., aBPIV-1, BPIV-2, or BPV-3 vector) or human PIV vector, a metapneumovirus(MPV) vector, a Sendia virus vector, or a measles virus vector, is usedto express a disclosed antigen. A BPIV3 viral vector expressing the RSVF and the hPIV F proteins (MEDI-534) is currently in clinical trials asa RSV vaccine. Examples of paramyxovirus (PIV) vector for expressingantigens are known to the person of skill in the art (see, e.g., U.S.Pat. App. Pubs. 2012/0045471, 2011/0212488, 2010/0297730, 2010/0278813,2010/0167270, 2010/0119547, 2009/0263883, 2009/0017517, 2009/0004722,2008/0096263, 2006/0216700, 2005/0147623, 2005/0142148, 2005/0019891,2004/0208895, 2004/0005545, 2003/0232061, 2003/0095987, and2003/0072773; each of which is incorporated by reference herein in itsentirety). In another example, a Newcastle disease viral vector is usedto express a disclosed antigen (see, e.g., McGinnes et al., J. Virol.,85: 366-377, 2011, describing RSV F and G proteins expressed onNewcastle disease like particles, incorporated by reference in itsentirety). In another example, a Sendai virus vector is used to expressa disclosed antigen (see, e.g., Jones et al., Vaccine, 30:959-968, 2012,incorporated by reference herein in its entirety, which discloses use ofa Sendai virus-based RSV vaccine to induce an immune response inprimates).

Additional viral vectors are also available for expression of thedisclosed antigens, including polyoma, i.e., SV40 (Madzak et al., 1992,J. Gen. Virol., 73:15331536), adenovirus (Berkner, 1992, Cur. Top.Microbiol. Immunol., 158:39-6; Berliner et al., 1988, Bio Techniques,6:616-629; Gorziglia et al., 1992, J. Virol., 66:4407-4412; Quantin etal., 1992, Proc. Natl. Acad. Sci. USA, 89:2581-2584; Rosenfeld et al.,1992, Cell, 68:143-155; Wilkinson et al., 1992, Nucl. Acids Res.,20:2233-2239; Stratford-Perricaudet et al., 1990, Hum. Gene Ther.,1:241-256), vaccinia virus (Mackett et al., 1992, Biotechnology,24:495-499), adeno-associated virus (Muzyczka, 1992, Curr. Top.Microbiol. Immunol., 158:91-123; On et al., 1990, Gene, 89:279-282),herpes viruses including HSV and EBV and CMV (Margolskee, 1992, Curr.Top. Microbiol. Immunol., 158:67-90; Johnson et al., 1992, J. Virol.,66:29522965; Fink et al., 1992, Hum. Gene Ther. 3:11-19; Breakfield etal., 1987, Mol. Neurobiol., 1:337-371; Fresse et al., 1990, Biochem.Pharmacol., 40:2189-2199), Sindbis viruses (H. Herweijer et al., 1995,Human Gene Therapy 6:1161-1167; U.S. Pat. No. 5,091,309 and U.S. Pat.No. 5,2217,879), alphaviruses (S. Schlesinger, 1993, Trends Biotechnol.11:18-22; I. Frolov et al., 1996, Proc. Natl. Acad. Sci. USA93:11371-11377) and retroviruses of avian (Brandyopadhyay et al., 1984,Mol. Cell. Biol., 4:749-754; Petropouplos et al., 1992, J. Virol.,66:3391-3397), murine (Miller, 1992, Curr. Top. Microbiol. Immunol.,158:1-24; Miller et al., 1985, Mol. Cell. Biol., 5:431-437; Sorge etal., 1984, Mol. Cell. Biol., 4:1730-1737; Mann et al., 1985, J. Virol.,54:401-407), and human origin (Page et al., 1990, J. Virol.,64:5370-5276; Buchschalcher et al., 1992, J. Virol., 66:2731-2739).Baculovirus (Autographa californica multinuclear polyhedrosis virus;AcMNPV) vectors are also known in the art, and may be obtained fromcommercial sources (such as PharMingen, San Diego, Calif.; ProteinSciences Corp., Meriden, Conn.; Stratagene, La Jolla, Calif.).Additional viral vectors are familiar to the person of ordinary skill inthe art.

In several embodiments, the methods and compositions disclosed hereininclude an adenoviral vector that expresses a recombinant RSV F proteinstabilized in a prefusion conformation. Adenovirus from various origins,subtypes, or mixture of subtypes can be used as the source of the viralgenome for the adenoviral vector. Non-human adenovirus (e.g., simian,chimpanzee, gorilla, avian, canine, ovine, or bovine adenoviruses) canbe used to generate the adenoviral vector. For example, a simianadenovirus can be used as the source of the viral genome of theadenoviral vector. A simian adenovirus can be of serotype 1, 3, 7, 11,16, 18, 19, 20, 27, 33, 38, 39, 48, 49, 50, or any other simianadenoviral serotype. A simian adenovirus can be referred to by using anysuitable abbreviation known in the art, such as, for example, SV, SAdV,SAV or sAV. In some examples, a simian adenoviral vector is a simianadenoviral vector of serotype 3, 7, 11, 16, 18, 19, 20, 27, 33, 38, or39. In one example, a chimpanzee serotype C Ad3 vector is used (see,e.g., Peruzzi et al., Vaccine, 27:1293-1300, 2009). Human adenovirus canbe used as the source of the viral genome for the adenoviral vector.Human adenovirus can be of various subgroups or serotypes. For instance,an adenovirus can be of subgroup A (e.g., serotypes 12, 18, and 31),subgroup B (e.g., serotypes 3, 7, 11, 14, 16, 21, 34, 35, and 50),subgroup C (e.g., serotypes 1, 2, 5, and 6), subgroup D (e.g., serotypes8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32,33, 36-39, and 42-48), subgroup E (e.g., serotype 4), subgroup F (e.g.,serotypes 40 and 41), an unclassified serogroup (e.g., serotypes 49 and51), or any other adenoviral serotype. The person of ordinary skill inthe art is familiar with replication competent and deficient adenoviralvectors (including singly and multiply replication deficient adenoviralvectors). Examples of replication-deficient adenoviral vectors,including multiply replication-deficient adenoviral vectors, aredisclosed in U.S. Pat. Nos. 5,837,511; 5,851,806; 5,994,106; 6,127,175;6,482,616; and 7,195,896, and International Patent Application Nos. WO94/28152, WO 95/02697, WO 95/16772, WO 95/34671, WO 96/22378, WO97/12986, WO 97/21826, and WO 03/02231 1.

E. Compositions

The disclosed PreF antigens, viral vectors, and nucleic acid moleculescan be included in a pharmaceutical composition, including therapeuticand prophylactic formulations, and can be combined together with one ormore adjuvants and, optionally, other therapeutic ingredients, such asantiviral drugs. In several embodiments, compositions including one ormore of the disclosed PreF antigens, viral vectors, or nucleic acidmolecules are immunogenic compositions.

Such pharmaceutical compositions can be administered to subjects by avariety of administration modes known to the person of ordinary skill inthe art, for example, nasal, pulmonary, intramuscular, subcutaneous,intravenous, intraperitoneal, or parenteral routes.

To formulate the compositions, the disclosed PreF antigens, viralvectors, or nucleic acid molecules can be combined with variouspharmaceutically acceptable additives, as well as a base or vehicle fordispersion of the conjugate. Desired additives include, but are notlimited to, pH control agents, such as arginine, sodium hydroxide,glycine, hydrochloric acid, citric acid, and the like. In addition,local anesthetics (for example, benzyl alcohol), isotonizing agents (forexample, sodium chloride, mannitol, sorbitol), adsorption inhibitors(for example, TWEEN® 80), solubility enhancing agents (for example,cyclodextrins and derivatives thereof), stabilizers (for example, serumalbumin), and reducing agents (for example, glutathione) can beincluded. Adjuvants, such as aluminum hydroxide (ALHYDROGEL®, availablefrom Brenntag Biosector, Copenhagen, Denmark and AMPHOGEL®, WyethLaboratories, Madison, N.J.), Freund's adjuvant, MPL™ (3-O-deacylatedmonophosphoryl lipid A; Corixa, Hamilton, Ind.), IL-12 (GeneticsInstitute, Cambridge, Mass.) TLR agonists (such as TLR-9 agonists),among many other suitable adjuvants well known in the art, can beincluded in the compositions.

When the composition is a liquid, the tonicity of the formulation, asmeasured with reference to the tonicity of 0.9% (w/v) physiologicalsaline solution taken as unity, is typically adjusted to a value atwhich no substantial, irreversible tissue damage will be induced at thesite of administration. Generally, the tonicity of the solution isadjusted to a value of about 0.3 to about 3.0, such as about 0.5 toabout 2.0, or about 0.8 to about 1.7.

The disclosed PreF antigens, viral vectors, or nucleic acid moleculescan be dispersed in a base or vehicle, which can include a hydrophiliccompound having a capacity to disperse the antigens, and any desiredadditives. The base can be selected from a wide range of suitablecompounds, including but not limited to, copolymers of polycarboxylicacids or salts thereof, carboxylic anhydrides (for example, maleicanhydride) with other monomers (for example, methyl (meth)acrylate,acrylic acid and the like), hydrophilic vinyl polymers, such aspolyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone, cellulosederivatives, such as hydroxymethylcellulose, hydroxypropylcellulose andthe like, and natural polymers, such as chitosan, collagen, sodiumalginate, gelatin, hyaluronic acid, and nontoxic metal salts thereof.Often, a biodegradable polymer is selected as a base or vehicle, forexample, polylactic acid, poly(lactic acid-glycolic acid) copolymer,polyhydroxybutyric acid, poly(hydroxybutyric acid-glycolic acid)copolymer and mixtures thereof. Alternatively or additionally, syntheticfatty acid esters such as polyglycerin fatty acid esters, sucrose fattyacid esters and the like can be employed as vehicles. Hydrophilicpolymers and other vehicles can be used alone or in combination, andenhanced structural integrity can be imparted to the vehicle by partialcrystallization, ionic bonding, cross-linking and the like. The vehiclecan be provided in a variety of forms, including fluid or viscoussolutions, gels, pastes, powders, microspheres and films, for examplesfor direct application to a mucosal surface.

The disclosed PreF antigens, viral vectors, or nucleic acid moleculescan be combined with the base or vehicle according to a variety ofmethods, and release of the antigens can be by diffusion, disintegrationof the vehicle, or associated formation of water channels. In somecircumstances, the disclosed antigens, or a nucleic acid or a viralvector encoding, expressing or including the antigen, is dispersed inmicrocapsules (microspheres) or nanocapsules (nanospheres) prepared froma suitable polymer, for example, isobutyl 2-cyanoacrylate (see, forexample, Michael et al., J. Pharmacy Pharmacol. 43:1-5, 1991), anddispersed in a biocompatible dispersing medium, which yields sustaineddelivery and biological activity over a protracted time.

The pharmaceutical compositions can contain as pharmaceuticallyacceptable vehicles substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents, wetting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, sorbitan monolaurate, and triethanolamine oleate. For solidcompositions, conventional nontoxic pharmaceutically acceptable vehiclescan be used which include, for example, pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum,cellulose, glucose, sucrose, magnesium carbonate, and the like.

Pharmaceutical compositions for administering the disclosed PreFantigens, viral vectors, or nucleic acid molecules can also beformulated as a solution, microemulsion, or other ordered structuresuitable for high concentration of active ingredients. The vehicle canbe a solvent or dispersion medium containing, for example, water,ethanol, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol, and the like), and suitable mixtures thereof.Proper fluidity for solutions can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of a desired particlesize in the case of dispersible formulations, and by the use ofsurfactants. In many cases, it will be desirable to include isotonicagents, for example, sugars, polyalcohols, such as mannitol andsorbitol, or sodium chloride in the composition. Prolonged absorption ofthe disclosed antigens can be brought about by including in thecomposition an agent which delays absorption, for example, monostearatesalts and gelatin.

In certain embodiments, the disclosed PreF antigens, viral vectors, ornucleic acid molecules can be administered in a time-releaseformulation, for example in a composition that includes a slow releasepolymer. These compositions can be prepared with vehicles that willprotect against rapid release, for example a controlled release vehiclesuch as a polymer, microencapsulated delivery system or bioadhesive gel.Prolonged delivery in various compositions of the disclosure can bebrought about by including in the composition agents that delayabsorption, for example, aluminum monostearate hydrogels and gelatin.When controlled release formulations are desired, controlled releasebinders suitable for use in accordance with the disclosure include anybiocompatible controlled release material which is inert to the activeagent and which is capable of incorporating the disclosed antigen and/orother biologically active agent. Numerous such materials are known inthe art. Useful controlled-release binders are materials that aremetabolized slowly under physiological conditions following theirdelivery (for example, at a mucosal surface, or in the presence ofbodily fluids). Appropriate binders include, but are not limited to,biocompatible polymers and copolymers well known in the art for use insustained release formulations. Such biocompatible compounds arenon-toxic and inert to surrounding tissues, and do not triggersignificant adverse side effects, such as nasal irritation, immuneresponse, inflammation, or the like. They are metabolized into metabolicproducts that are also biocompatible and easily eliminated from thebody. Numerous systems for controlled delivery of therapeutic proteinsare known (e.g., U.S. Pat. No. 5,055,303; U.S. Pat. No. 5,188,837; U.S.Pat. No. 4,235,871; U.S. Pat. No. 4,501,728; U.S. Pat. No. 4,837,028;U.S. Pat. No. 4,957,735; and U.S. Pat. No. 5,019,369; U.S. Pat. No.5,055,303; U.S. Pat. No. 5,514,670; U.S. Pat. No. 5,413,797; U.S. Pat.No. 5,268,164; U.S. Pat. No. 5,004,697; U.S. Pat. No. 4,902,505; U.S.Pat. No. 5,506,206; U.S. Pat. No. 5,271,961; U.S. Pat. No. 5,254,342;and U.S. Pat. No. 5,534,496).

Exemplary polymeric materials for use include, but are not limited to,polymeric matrices derived from copolymeric and homopolymeric polyestershaving hydrolyzable ester linkages. A number of these are known in theart to be biodegradable and to lead to degradation products having no orlow toxicity. Exemplary polymers include polyglycolic acids andpolylactic acids, poly(DL-lactic acid-co-glycolic acid), poly(D-lacticacid-co-glycolic acid), and poly(L-lactic acid-co-glycolic acid). Otheruseful biodegradable or bioerodible polymers include, but are notlimited to, such polymers as poly(epsilon-caprolactone),poly(epsilon-aprolactone-CO-lactic acid),poly(epsilon.-aprolactone-CO-glycolic acid), poly(beta-hydroxy butyricacid), poly(alkyl-2-cyanoacrylate), hydrogels, such as poly(hydroxyethylmethacrylate), polyamides, poly(amino acids) (for example, L-leucine,glutamic acid, L-aspartic acid and the like), poly(ester urea),poly(2-hydroxyethyl DL-aspartamide), polyacetal polymers,polyorthoesters, polycarbonate, polymaleamides, polysaccharides, andcopolymers thereof. Many methods for preparing such formulations arewell known to those skilled in the art (see, for example, Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978). Other useful formulations includecontrolled-release microcapsules (U.S. Pat. Nos. 4,652,441 and4,917,893), lactic acid-glycolic acid copolymers useful in makingmicrocapsules and other formulations (U.S. Pat. Nos. 4,677,191 and4,728,721) and sustained-release compositions for water-soluble peptides(U.S. Pat. No. 4,675,189).

Pharmaceutical compositions typically are sterile and stable underconditions of manufacture, storage and use. Sterile solutions can beprepared by incorporating the conjugate in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedherein, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the disclosed antigen and/orother biologically active agent into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated herein. In the case of sterile powders, methods ofpreparation include vacuum drying and freeze-drying which yields apowder of the disclosed antigen plus any additional desired ingredientfrom a previously sterile-filtered solution thereof. The prevention ofthe action of microorganisms can be accomplished by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

Actual methods for preparing administrable compositions will be known orapparent to those skilled in the art and are described in more detail insuch publications as Remingtons Pharmaceutical Sciences, 19^(th) Ed.,Mack Publishing Company, Easton, Pa., 1995.

In several embodiments, the compositions include an adjuvant. The personof ordinary skill in the art is familiar with adjuvants, for example,those that can be included in an immunogenic composition. In severalembodiments, the adjuvant is selected to elicit a Th1 biased immuneresponse in a subject administered an immunogenic composition containingthe adjuvant and a disclosed antigens, or a nucleic acid or a viralvector encoding, expressing or including the antigen.

One suitable adjuvant is a non-toxic bacterial lipopolysaccharidederivative. An example of a suitable non-toxic derivative of lipid A, ismonophosphoryl lipid A or more particularly 3-Deacylated monophosphoryllipid A (3D-MPL). See, for example, U.S. Pat. Nos. 4,436,727; 4,877,611;4,866,034 and 4,912,094. 3D-MPL primarily promotes CD4+ T cell responseswith an IFN-γ (Thl) phenotype. 3D-MPL can be produced according to themethods disclosed in GB2220211 A. Chemically it is a mixture of3-deacylated monophosphoryl lipid A with 3, 4, 5 or 6 acylated chains.In the compositions, small particle 3D-MPL can be used. Small particle3D-MPL has a particle size such that it can be sterile-filtered througha 0.22 μm filter. Such preparations are described in WO94/21292.

In other embodiments, the lipopolysaccharide can be a β(1-6) glucosaminedisaccharide, as described in U.S. Pat. No. 6,005,099 and EP Patent No.0 729 473 Bl. One of skill in the art would be readily able to producevarious lipopolysaccharides, such as 3D-MPL, based on the teachings ofthese references. In addition to the aforementioned immunostimulants(that are similar in structure to that of LPS or MPL or 3D-MPL),acylated monosaccharide and disaccharide derivatives that are asub-portion to the above structure of MPL are also suitable adjuvants.

In several embodiments, a Toll-like receptor (TLR) agonist is used as anadjuvant. For example a disclosed PreF antigen can be combined with aTLR agonist in an immunogenic composition used for elicitation of aneutralizing immune response to RSV. For example, the TLR agonist can bea TLR-4 agonist such as a synthetic derivative of lipid A (see, e.g., WO95/14026, and WO 01/46127) an alkyl Glucosaminide phosphate (AGP; see,e.g., WO 98/50399 or U.S. Pat. Nos. 6,303,347; 6,764,840). Othersuitable TLR-4 ligands, capable of causing a signaling response throughTLR-4 are, for example, lipopolysaccharide from gram-negative bacteriaand its derivatives, or fragments thereof, in particular a non-toxicderivative of LPS (such as 3D-MPL). Other suitable TLR agonists are:heat shock protein (HSP) 10, 60, 65, 70, 75 or 90; surfactant Protein A,hyaluronan oligosaccharides, heparan sulphate fragments, fibronectinfragments, fibrinogen peptides and β-defensin-2, and muramyl dipeptide(MDP). In one embodiment the TLR agonist is HSP 60, 70 or 90. Othersuitable TLR-4 ligands are as described in WO 2003/011223 and in WO2003/099195.

Additional TLR agonists (such as an agent that is capable of causing asignaling response through a TLR signaling pathway) are also useful asadjuvants, such as agonists for TLR2, TLR3, TLR7, TLR8 and/or TLR9.Accordingly, in one embodiment, the composition further includes anadjuvant which is selected from the group consisting of: a TLR-1agonist, a TLR-2 agonist, TLR-3 agonist, a TLR-4 agonist, TLR-5 agonist,a TLR-6 agonist, TLR-7 agonist, a TLR-8 agonist, TLR-9 agonist, or acombination thereof.

In one embodiment, a TLR agonist is used that is capable of causing asignaling response through TLR-1, for example one or more of from:Tri-acylated lipopeptides (LPs); phenol-soluble modulin; Mycobacteriumtuberculosis LP;S-(2,3-bis(palmitoyloxy)-(2-RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser-(S)-L-ys(4)-OH,trihydrochloride (Pam3Cys) LP which mimics the acetylated amino terminusof a bacterial lipoprotein and OspA LP from Borrelia burgdorferi. Inanother embodiment, a TLR agonist is used that is capable of causing asignaling response through TLR-2, such as one or more of a lipoprotein,a peptidoglycan, a bacterial lipopeptide from M. tuberculosis, B.burgdorferi or T. pallidum; peptidoglycans from species includingStaphylococcus aureus; lipoteichoic acids, mannuronic acids, Neisseriaporins, bacterial fimbriae, Yersina virulence factors, CMV virions,measles hemagglutinin, and zymosan from yeast. In some embodiments, aTLR agonist is used that is capable of causing a signaling responsethrough TLR-3, such as one or more of double stranded RNA (dsRNA), orpolyinosinic-polycytidylic acid (Poly IC), a molecular nucleic acidpattern associated with viral infection. In further embodiments, a TLRagonist is used that is capable of causing a signaling response throughTLR-5, such as bacterial flagellin. In additional embodiments, a TLRagonist is used that is capable of causing a signaling response throughTLR-6, such as one or more of mycobacterial lipoprotein, di-acylated LP,and phenol-soluble modulin. Additional TLR6 agonists are described in WO2003/043572. In an embodiment, a TLR agonist is used that is capable ofcausing a signaling response through TLR-7, such as one or more of asingle stranded RNA (ssRNA), loxoribine, a guanosine analogue atpositions N7 and C8, or an imidazoquinoline compound, or derivativethereof. In one embodiment, the TLR agonist is imiquimod. Further TLR7agonists are described in WO 2002/085905. In some embodiments, a TLRagonist is used that is capable of causing a signaling response throughTLR-8. Suitably, the TLR agonist capable of causing a signaling responsethrough TLR-8 is a single stranded RNA (ssRNA), an imidazoquinolinemolecule with anti-viral activity, for example resiquimod (R848);resiquimod is also capable of recognition by TLR-7. Other TLR-8 agonistswhich can be used include those described in WO 2004/071459.

In further embodiments, an adjuvant includes a TLR agonist capable ofinducing a signaling response through TLR-9. For example, the adjuvantcan include HSP90, bacterial or viral DNA, and/or DNA containingunmethylated CpG nucleotides (e.g., a CpG oligonucleotide). For example,CpG-containing oligonucleotides induce a predominantly Th1 response.Such oligonucleotides are well known and are described, for example, inWO 95/26204, WO 96/02555, WO 99/33488 and U.S. Pat. Nos. 5,278,302,5,666,153, and U.S. Pat. No. 6,008,200 and U.S. Pat. No. 5,856,462.Accordingly, oligonucleotides for use as adjuvants in the disclosedcompositions include CpG containing oligonucleotides, for example,containing two or more dinucleotide CpG motifs. Also included areoligonucleotides with mixed internucleotide linkages.

Other adjuvants that can be used in immunogenic compositions with theantigens, or a nucleic acid or a viral vector encoding, expressing orincluding an antigen, e.g., on their own or in combination with 3D-MPL,or another adjuvant described herein, are saponins, such as QS21. Insome examples, saponins are used as an adjuvant, e.g., for systemicadministration of a PreF antigen. Use of saponins (e.g., use of Quil A,derived from the bark of the South American tree Quillaja SaponariaMolina) as adjuvants is familiar to the person of ordinary skill in theart (see, e.g., U.S. Pat. No. 5,057,540 and EP 0 362 279 Bl. EP 0 109942 B1; WO 96/11711; WO 96/33739). The haemolytic saponins QS21 and QS17(HPLC purified fractions of Quil A) have been described as potentsystemic adjuvants, and the method of their production is disclosed inU.S. Pat. No. 5,057,540 and EP 0 362 279 B1.

The adjuvant can also include mineral salts such as an aluminum orcalcium salts, in particular aluminum hydroxide, aluminum phosphate andcalcium phosphate.

Another class of suitable Th1 biasing adjuvants for use in compositionsincludes outer membrane proteins (OMP)-based immunostimulatorycompositions. OMP-based immunostimulatory compositions are particularlysuitable as mucosal adjuvants, e.g., for intranasal administration.OMP-based immunostimulatory compositions are a genus of preparations of(OMPs, including some porins) from Gram-negative bacteria, e.g.,Neisseria species, which are useful as a carrier or in compositions forimmunogens, such as bacterial or viral antigens (see, e.g., U.S. Pat.No. 5,726,292; U.S. Pat. No. 4,707,543). Further, proteosomes have thecapability to auto-assemble into vesicle or vesicle-like OMP clusters ofabout 20 nm to about 800 nm, and to noncovalently incorporate,coordinate, associate (e.g., electrostatically or hydrophobically), orotherwise cooperate with protein antigens (Ags), particularly antigensthat have a hydrophobic moiety. Proteosomes can be prepared, forexample, as described in the art (see, e.g., U.S. Pat. No. 5,726,292 orU.S. Pat. No. 5,985,284; 2003/0044425.).

Proteosomes are composed primarily of chemically extracted outermembrane proteins (OMP5) from Neisseria meningitidis (mostly porins Aand B as well as class 4 OMP), maintained in solution by detergent(Lowell G H. Proteosomes for Improved Nasal, Oral, or InjectableVaccines. In: Levine M M, Woodrow G C, Kaper J B, Cobon G S, eds, NewGeneration Vaccines. New York: Marcel Dekker, Inc. 1997; 193-206).Proteosomes can be formulated with a variety of antigens such aspurified or recombinant proteins derived from viral sources, includingthe PreF polypeptides disclosed herein. The gradual removal of detergentallows the formation of particulate hydrophobic complexes ofapproximately 100-200 nm in diameter (Lowell G H. Proteosomes forImproved Nasal, Oral, or Injectable Vaccines. In: Levine M M, Woodrow GC, Kaper J B, Cobon G S, eds, New Generation Vaccines. New York: MarcelDekker, Inc. 1997; 193-206).

Combinations of different adjuvants can also be used in compositionswith the disclosed PreF antigens, viral vectors, or nucleic acidmolecules in the composition. For example, as already noted, QS21 can beformulated together with 3D-MPL. The ratio of QS21:3 D-MPL willtypically be in the order of 1:10 to 10:1; such as 1:5 to 5:1, and oftensubstantially 1:1. Typically, the ratio is in the range of 2.5:1 to 1:13D-MPL:QS21 (such as AS01 (GlaxoSmithKline). Another combinationadjuvant formulation includes 3D-MPL and an aluminum salt, such asaluminum hydroxide (such as AS04 (GlaxoSmithKline). When formulated incombination, this combination can enhance an antigen-specific Thl immuneresponse.

In some instances, the adjuvant formulation a mineral salt, such as acalcium or aluminum (alum) salt, for example calcium phosphate, aluminumphosphate or aluminum hydroxide. In some embodiments, the adjuvantincludes an oil and water emulsion, e.g., an oil-in-water emulsion (suchas MF59 (Novartis) or AS03 (GlaxoSmithKline). One example of anoil-in-water emulsion comprises a metabolizable oil, such as squalene, atocol such as a tocopherol, e.g., alpha-tocopherol, and a surfactant,such as sorbitan trioleate (Span 85) or polyoxyethylene sorbitanmonooleate (Tween 80), in an aqueous carrier.

The pharmaceutical composition typically contains a therapeuticallyeffective amount of a disclosed PreF antigen, viral vector, or nucleicacid molecule and can be prepared by conventional techniques.Preparation of immunogenic compositions, including those foradministration to human subjects, is generally described inPharmaceutical Biotechnology, Vol. 61 Vaccine Design—the subunit andadjuvant approach, edited by Powell and Newman, Plenum Press, 1995. NewTrends and Developments in Vaccines, edited by Voller et al., UniversityPark Press, Baltimore, Md., U.S.A. 1978. Encapsulation within liposomesis described, for example, by Fullerton, U.S. Pat. No. 4,235,877.Conjugation of proteins to macromolecules is disclosed, for example, byLikhite, U.S. Pat. No. 4,372,945 and by Armor et al., U.S. Pat. No.4,474,757. Typically, the amount of antigen in each dose of theimmunogenic composition is selected as an amount which induces an immuneresponse without significant, adverse side effects.

The amount of the disclosed PreF antigen, viral vector, or nucleic acidmolecule can vary depending upon the specific antigen employed, theroute and protocol of administration, and the target population, forexample. Typically, each human dose will comprise 1-1000 μg of protein,such as from about 1 μg to about 100 μg, for example, from about 1 μg toabout 50 μg, such as about 1 μg, about 2 μg, about 5 μg, about 10 μg,about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 40 μg, orabout 50 μg. The amount utilized in an immunogenic composition isselected based on the subject population (e.g., infant or elderly). Anoptimal amount for a particular composition can be ascertained bystandard studies involving observation of antibody titers and otherresponses in subjects. It is understood that a therapeutically effectiveamount of an antigen in a immunogenic composition can include an amountthat is ineffective at eliciting an immune response by administration ofa single dose, but that is effective upon administration of multipledosages, for example in a prime-boost administration protocol.

In several examples, pharmaceutical compositions for eliciting an immuneresponse against RSV in humans include a therapeutically effectiveamount of a disclosed PreF antigens, viral vectors, or nucleic acidmolecules for administration to infants (e.g., infants between birth and1 year, such as between 0 and 6 months, at the age of initial dose) orelderly patients subject (such as a subject greater than 65 years ofage). It will be appreciated that the choice of adjuvant can bedifferent in these different applications, and the optimal adjuvant andconcentration for each situation can be determined empirically by thoseof skill in the art.

In certain embodiments, the pharmaceutical compositions are vaccinesthat reduce or prevent infection with RSV. In some embodiments, theimmunogenic compositions are vaccines that reduce or prevent apathological response following infection with RSV. Optionally, thepharmaceutical compositions containing the disclosed PreF antigen, viralvector, or nucleic acid molecule are formulated with at least oneadditional antigen of a pathogenic organism other than RSV. For example,the pathogenic organism can be a pathogen of the respiratory tract (suchas a virus or bacterium that causes a respiratory infection). In certaincases, the pharmaceutical composition contains an antigen derived from apathogenic virus other than RSV, such as a virus that causes aninfection of the respiratory tract, such as influenza or parainfluenza.In other embodiments, the additional antigens are selected to facilitateadministration or reduce the number of inoculations required to protecta subject against a plurality of infectious organisms. For example, theantigen can be derived from any one or more of influenza, hepatitis B,diphtheria, tetanus, pertussis, Hemophilus influenza, poliovirus,Streptococcus or Pneumococcus, among others.

F. Methods of Treatment

In several embodiments, the disclosed PreF antigens, or a nucleic acidor a viral vector encoding, expressing or including a PreF antigen areused to induce an immune response to RSV in a subject. Thus, in severalembodiments, a therapeutically effective amount of an immunogeniccomposition including one or more of the disclosed PreF antigens, or anucleic acid or a viral vector encoding, expressing or including theantigen, can be administered to a subject in order to generate an immuneresponse to RSV.

In accordance with the disclosure herein, a prophylactically ortherapeutically effective amount of a immunogenic composition includinga PreF antigen, or a nucleic acid or a viral vector encoding, expressingor including the antigen, is administered to a subject in need of suchtreatment for a time and under conditions sufficient to prevent,inhibit, and/or ameliorate a RSV infection in a subject. The immunogeniccomposition is administered in an amount sufficient to elicit an immuneresponse against an RSV antigen, such as RSV F protein, in the subject.

In some embodiments, a subject is selected for treatment that has, or isat risk for developing, an RSV infection, for example, because ofexposure or the possibility of exposure to RSV. Following administrationof a therapeutically effective amount of the disclosed therapeuticcompositions, the subject can be monitored for RSV infection, symptomsassociated with RSV infection, or both. Because nearly all humans areinfected with RSV by the age of 3, the entire birth cohort is includedas a relevant population for immunization. This could be done, forexample, by beginning an immunization regimen anytime from birth to 6months of age, from 6 months of age to 5 years of age, in pregnant women(or women of child-bearing age) to protect their infants by passivetransfer of antibody, family members of newborn infants or those stillin utero, and subjects greater than 50 years of age.

Subjects at greatest risk of RSV infection with severe symptoms (e.g.requiring hospitalization) include children with prematurity,bronchopulmonary dysplasia, and congenital heart disease are mostsusceptible to severe disease. Atopy or a family history of atopy hasalso been associated with severe disease in infancy. During childhoodand adulthood, disease is milder but can be associated with lower airwaydisease and is commonly complicated by sinusitis. Disease severityincreases in the institutionalized elderly (e.g., humans over 65 yearsold). Severe disease also occurs in persons with severe combinedimmunodeficiency disease or following bone marrow or lungtransplantation. (See, e.g., Shay et al., JAMA, 282:1440-6, 1999; Hallet al., N Engl J. Med. 2009; 360:588-598; Glezen et al., Am J DisChild., 1986; 140:543-546; and Graham, Immunol. Rev., 239:149-166, 2011,each of which is incorporated by reference herein). Thus, these subjectscan be selected for administration of the disclosed PreF antigens, or anucleic acid or a viral vector encoding, expressing or including a PreFantigen.

Typical subjects intended for treatment with the compositions andmethods of the present disclosure include humans, as well as non-humanprimates and other animals, such as cattle. To identify subjects forprophylaxis or treatment according to the methods of the disclosure,screening methods employed to determine risk factors associated with atargeted or suspected disease or condition, or to determine the statusof an existing disease or condition in a subject. These screeningmethods include, for example, conventional work-ups to determineenvironmental, familial, occupational, and other such risk factors thatmay be associated with the targeted or suspected disease or condition,as well as diagnostic methods, such as various ELISA and otherimmunoassay methods, which are available and well known in the art todetect and/or characterize RSV infection. These and other routinemethods allow the clinician to select patients in need of therapy usingthe methods and pharmaceutical compositions of the disclosure. Animmunogenic composition can be administered as an independentprophylaxis or treatment program, or as a follow-up, adjunct orcoordinate treatment regimen to other treatments.

The immunogenic composition can be used in coordinate vaccinationprotocols or combinatorial formulations. In certain embodiments,combinatorial immunogenic compositions and coordinate immunizationprotocols employ separate immunogens or formulations, each directedtoward eliciting an immune response to an RSV antigen, such as an immuneresponse to RSV F protein. Separate immunogenic compositions that elicitthe immune response to the RSV antigen can be combined in a polyvalentimmunogenic composition administered to a subject in a singleimmunization step, or they can be administered separately (in monovalentimmunogenic compositions) in a coordinate immunization protocol.

The administration of the immunogenic compositions can be for eitherprophylactic or therapeutic purpose. When provided prophylactically, theimmunogenic composition is provided in advance of any symptom, forexample in advance of infection. The prophylactic administration of theimmunogenic compositions serves to prevent or ameliorate any subsequentinfection. When provided therapeutically, the immunogenic compositionsare provided at or after the onset of a symptom of disease or infection,for example after development of a symptom of RSV infection, or afterdiagnosis of RSV infection. The immunogenic composition can thus beprovided prior to the anticipated exposure to RSV so as to attenuate theanticipated severity, duration or extent of an infection and/orassociated disease symptoms, after exposure or suspected exposure to thevirus, or after the actual initiation of an infection.

Administration induces a sufficient immune response to treat or preventthe pathogenic infection, for example, to inhibit the infection and/orreduce the signs and/or symptoms of the infection. Amounts effective forthis use will depend upon the severity of the disease, the general stateof the subject's health, and the robustness of the subject's immunesystem. A therapeutically effective amount of the disclosed immunogeniccompositions is that which provides either subjective relief of asymptom(s) or an objectively identifiable improvement as noted by theclinician or other qualified observer.

For prophylactic and therapeutic purposes, the immunogenic compositioncan be administered to the subject in a single bolus delivery, viacontinuous delivery (for example, continuous transdermal, mucosal orintravenous delivery) over an extended time period, or in a repeatedadministration protocol (for example, by an hourly, daily or weekly,repeated administration protocol). The therapeutically effective dosageof the immunogenic composition can be provided as repeated doses withina prolonged prophylaxis or treatment regimen that will yield clinicallysignificant results to alleviate one or more symptoms or detectableconditions associated with a targeted disease or condition as set forthherein. Determination of effective dosages in this context is typicallybased on animal model studies followed up by human clinical trials andis guided by administration protocols that significantly reduce theoccurrence or severity of targeted disease symptoms or conditions in thesubject. Suitable models in this regard include, for example, murine,rat, porcine, feline, ferret, non-human primate, and other acceptedanimal model subjects known in the art. Alternatively, effective dosagescan be determined using in vitro models (for example, immunologic andhistopathologic assays). Using such models, only ordinary calculationsand adjustments are required to determine an appropriate concentrationand dose to administer a therapeutically effective amount of theimmunogenic composition (for example, amounts that are effective toelicit a desired immune response or alleviate one or more symptoms of atargeted disease). In alternative embodiments, an effective amount oreffective dose of the immunogenic composition may simply inhibit orenhance one or more selected biological activities correlated with adisease or condition, as set forth herein, for either therapeutic ordiagnostic purposes.

In one embodiment, a suitable immunization regimen includes at leastthree separate inoculations with one or more immunogenic compositions,with a second inoculation being administered more than about two, aboutthree to eight, or about four, weeks following the first inoculation.Generally, the third inoculation is administered several months afterthe second inoculation, and in specific embodiments, more than aboutfive months after the first inoculation, more than about six months toabout two years after the first inoculation, or about eight months toabout one year after the first inoculation. Periodic inoculations beyondthe third are also desirable to enhance the subject's “immune memory.”The adequacy of the vaccination parameters chosen, e.g., formulation,dose, regimen and the like, can be determined by taking aliquots ofserum from the subject and assaying antibody titers during the course ofthe immunization program. If such monitoring indicates that vaccinationis sub-optimal, the subject can be boosted with an additional dose ofimmunogenic composition, and the vaccination parameters can be modifiedin a fashion expected to potentiate the immune response. It iscontemplated that there can be several boosts, and that each boost caninclude the same or a different PreF antigen.

For prime-boost protocols, the prime can be administered as a singledose or multiple doses, for example two doses, three doses, four doses,five doses, six doses or more can be administered to a subject overdays, weeks or months. The boost can be administered as a single dose ormultiple doses, for example two to six doses, or more can beadministered to a subject over a day, a week or months. Multiple boostscan also be given, such one to five, or more. Different dosages can beused in a series of sequential inoculations. For example a relativelylarge dose in a primary inoculation and then a boost with relativelysmaller doses. The immune response against the selected antigenicsurface can be generated by one or more inoculations of a subject withan immunogenic composition disclosed herein.

The actual dosage of the immunogenic composition will vary according tofactors such as the disease indication and particular status of thesubject (for example, the subject's age, size, fitness, extent ofsymptoms, susceptibility factors, and the like), time and route ofadministration, other drugs or treatments being administeredconcurrently, as well as the specific pharmacology of the immunogeniccomposition for eliciting the desired activity or biological response inthe subject. Dosage regimens can be adjusted to provide an optimumprophylactic or therapeutic response. As described above in the forgoinglisting of terms, an effective amount is also one in which any toxic ordetrimental side effects of the disclosed antigen and/or otherbiologically active agent is outweighed in clinical terms bytherapeutically beneficial effects.

A non-limiting range for a therapeutically effective amount of thedisclosed PreF antigens within the methods and immunogenic compositionsof the disclosure is about 0.0001 mg/kg body weight to about 10 mg/kgbody weight, such as about 0.01 mg/kg, about 0.02 mg/kg, about 0.03mg/kg, about 0.04 mg/kg, about 0.05 mg/kg, about 0.06 mg/kg, about 0.07mg/kg, about 0.08 mg/kg, about 0.09 mg/kg, about 0.1 mg/kg, about 0.2mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.6mg/kg, about 0.7 mg/kg, about 0.8 mg/kg, about 0.9 mg/kg, about 1 mg/kg,about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 4mg/kg, about 5 mg/kg, or about 10 mg/kg, for example 0.01 mg/kg to about1 mg/kg body weight, about 0.05 mg/kg to about 5 mg/kg body weight,about 0.2 mg/kg to about 2 mg/kg body weight, or about 1.0 mg/kg toabout 10 mg/kg body weight.

In some embodiments, the dosage a set amount of a disclosed PreFantigen, or a nucleic acid or a viral vector encoding, expressing orincluding a PreF antigen includes for children, adults, elderly, etc.,such as from about 1-300 μg, for example, a dosage of about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200,250, or about 300 μg of the PreF antigens, or a nucleic acid or a viralvector encoding, expressing or including a PreF antigen. The dosage andnumber of doses will depend on the setting, for example, in an adult oranyone primed by prior RSV infection or immunization, a single dose maybe a sufficient booster. In naïve infants, in some examples, at leasttwo doses would be given, for example, at least three doses. In someembodiments, an annual boost is given to elderly subjects (e.g., humansover age 60) once per year, for example, along with an annual influenzavaccination. Methods for preparing administrable compositions will beknown or apparent to those skilled in the art and are described in moredetail in such publications as Remingtons Pharmaceutical Sciences,19^(th) Ed., Mack Publishing Company, Easton, Pa., 1995.

Dosage can be varied by the attending clinician to maintain a desiredconcentration at a target site (for example, systemic circulation).Higher or lower concentrations can be selected based on the mode ofdelivery, for example, trans-epidermal, rectal, oral, pulmonary, orintranasal delivery versus intravenous or subcutaneous delivery. Dosagecan also be adjusted based on the release rate of the administeredformulation, for example, of an intrapulmonary spray versus powder,sustained release oral versus injected particulate or transdermaldelivery formulations, and so forth. To achieve the same serumconcentration level, for example, slow-release particles with a releaserate of 5 nanomolar (under standard conditions) would be administered atabout twice the dosage of particles with a release rate of 10 nanomolar.

Upon administration of an immunogenic composition of this disclosure,the immune system of the subject typically responds to the immunogeniccomposition by producing antibodies specific for the prefusionconformation of the RSV F protein. Such a response signifies that aneffective dose of the immunogenic composition was delivered.

In several embodiments, it may be advantageous to administer theimmunogenic compositions disclosed herein with other agents such asproteins, peptides, antibodies, and other antiviral agents, such asanti-RSV agents. Non-limiting examples of anti-RSV agents include themonoclonal antibody palivizumab (SYNAGIS®; Medimmune, Inc.) and thesmall molecule anti-viral drug ribavirin (manufactured by many sources,e.g., Warrick Pharmaceuticals, Inc.). In certain embodiments,immunogenic compositions are administered concurrently with otheranti-RSV agents. In certain embodiments, the immunogenic compositionsare administered sequentially with other anti-RSV therapeutic agents,such as before or after the other agent. One of ordinary skill in theart would know that sequential administration can mean immediatelyfollowing or after an appropriate period of time, such as hours, days,weeks, months, or even years later.

In additional embodiments, a therapeutically effective amount of apharmaceutical composition including a nucleic acid encoding a disclosedPreF antigen is administered to a subject in order to generate an immuneresponse. In one specific, non-limiting example, a therapeuticallyeffective amount of a nucleic acid encoding a disclosed antigen isadministered to a subject to treat or prevent or inhibit RSV infection.

One approach to administration of nucleic acids is direct immunizationwith plasmid DNA, such as with a mammalian expression plasmid. Asdescribed above, the nucleotide sequence encoding a disclosed antigencan be placed under the control of a promoter to increase expression ofthe molecule. Another approach would use RNA (such as Nonviral deliveryof self-amplifying RNA vaccines, see e.g., Geall et al., Proc Natl AcadSci USA, 109:14604-9, 2012.

Immunization by nucleic acid constructs is well known in the art andtaught, for example, in U.S. Pat. No. 5,643,578 (which describes methodsof immunizing vertebrates by introducing DNA encoding a desired antigento elicit a cell-mediated or a humoral response), and U.S. Pat. No.5,593,972 and U.S. Pat. No. 5,817,637 (which describe operably linking anucleic acid sequence encoding an antigen to regulatory sequencesenabling expression). U.S. Pat. No. 5,880,103 describes several methodsof delivery of nucleic acids encoding immunogenic peptides or otherantigens to an organism. The methods include liposomal delivery of thenucleic acids (or of the synthetic peptides themselves), andimmune-stimulating constructs, or ISCOMS™, negatively charged cage-likestructures of 30-40 nm in size formed spontaneously on mixingcholesterol and Quil A™ (saponin). Protective immunity has beengenerated in a variety of experimental models of infection, includingtoxoplasmosis and Epstein-Barr virus-induced tumors, using ISCOMS™ asthe delivery vehicle for antigens (Mowat and Donachie, Immunol. Today12:383, 1991). Doses of antigen as low as 1 μg encapsulated in ISCOMS™have been found to produce Class I mediated CTL responses (Takahashi etal., Nature 344:873, 1990).

In another approach to using nucleic acids for immunization, a disclosedantigen can also be expressed by attenuated viral hosts or vectors orbacterial vectors. Recombinant vaccinia virus, adenovirus,adeno-associated virus (AAV), herpes virus, retrovirus, cytomegalovirusor other viral vectors can be used to express the peptide or protein,thereby eliciting a CTL response. For example, vaccinia vectors andmethods useful in immunization protocols are described in U.S. Pat. No.4,722,848. BCG (Bacillus Calmette Guerin) provides another vector forexpression of the peptides (see Stover, Nature 351:456-460, 1991).

In one embodiment, a nucleic acid encoding a disclosed PreF antigen isintroduced directly into cells. For example, the nucleic acid can beloaded onto gold microspheres by standard methods and introduced intothe skin by a device such as Bio-Rad's HELIOS™ Gene Gun. The nucleicacids can be “naked,” consisting of plasmids under control of a strongpromoter. Typically, the DNA is injected into muscle, although it canalso be injected directly into other sites, including tissues inproximity to metastases. Dosages for injection are usually around 0.5μg/kg to about 50 mg/kg, and typically are about 0.005 mg/kg to about 5mg/kg (see, e.g., U.S. Pat. No. 5,589,466).

In addition to the therapeutic methods provided above, any of thedisclosed PreF antigens can be utilized to produce antigen specificimmunodiagnostic reagents, for example, for serosurveillance.Immunodiagnostic reagents can be designed from any of the antigensdescribed herein. For example, in the case of the disclosed antigens,the presence of serum antibodies to RSV is monitored using the isolatedantigens disclosed herein, such as to detect an RSV infection and/or thepresence of antibodies that specifically bind to the prefusionconformation of RSV F protein.

Generally, the method includes contacting a sample from a subject, suchas, but not limited to a blood, serum, plasma, urine or sputum samplefrom the subject with one or more of the RSV F protein antigenstabilized in a prefusion conformation disclosed herein and detectingbinding of antibodies in the sample to the disclosed immunogens. Thebinding can be detected by any means known to one of skill in the art,including the use of labeled secondary antibodies that specifically bindthe antibodies from the sample. Labels include radiolabels, enzymaticlabels, and fluorescent labels.

In addition, the detection of the prefusion RSV F binding antibody alsoallows the response of the subject to immunization with the disclosedantigen to be monitored. In still other embodiments, the titer of theprefusion RSV F antibody binding antibodies is determined. The bindingcan be detected by any means known to one of skill in the art, includingthe use of labeled secondary antibodies that specifically bind theantibodies from the sample. Labels include radiolabels, enzymaticlabels, and fluorescent labels. In other embodiments, a disclosedimmunogen is used to isolate antibodies present in a subject orbiological sample obtained from a subject.

G. Kits

Kits are also provided. For example, kits for treating or preventing anRSV infection in a subject, or for detecting the presence of RSV Fprotein prefusion specific antibodies in the sera of a subject. The kitswill typically include one or more of the PreF antigens, or a nucleicacid or a viral vector encoding, expressing or including the antigen.

The kit can include a container and a label or package insert on orassociated with the container. Suitable containers include, for example,bottles, vials, syringes, etc. The containers may be formed from avariety of materials such as glass or plastic. The container typicallyholds a composition including one or more of the disclosed PreFantigens, or a nucleic acid or a viral vector encoding, expressing orincluding the antigen, which is effective for treating or preventing RSVinfection. In several embodiments the container may have a sterileaccess port (for example the container may be an intravenous solutionbag or a vial having a stopper pierceable by a hypodermic injectionneedle). The label or package insert indicates that the composition isused for treating the particular condition.

The label or package insert typically will further include instructionsfor use of a PreF antigen, or a nucleic acid or a viral vector encoding,expressing or including the antigen, for example, in a method oftreating or preventing a RSV infection. The package insert typicallyincludes instructions customarily included in commercial packages oftherapeutic products that contain information about the indications,usage, dosage, administration, contraindications and/or warningsconcerning the use of such therapeutic products. The instructionalmaterials may be written, in an electronic form (such as a computerdiskette or compact disk) or may be visual (such as video files). Thekits may also include additional components to facilitate the particularapplication for which the kit is designed. The kits may additionallyinclude buffers and other reagents routinely used for the practice of aparticular method. Such kits and appropriate contents are well known tothose of skill in the art.

H. Certain Embodiments

Additional embodiments are disclosed in section H on pages 87-114 ofpriority U.S. Provisional application No. 61/798,389, filed Mar. 15,2013, which is specifically incorporated by reference herein in itsentirety.

III. EXAMPLES

The following examples are provided to illustrate particular features ofcertain embodiments, but the scope of the claims should not be limitedto those features exemplified.

Example 1 Structure of Respiratory Syncytial Virus Prefusion F TrimerBound to a Human Antibody

The prefusion conformation of the respiratory syncytial virus (RSV)fusion (F) glycoprotein is the target of most RSV-neutralizingantibodies in human sera, but its metastability has hinderedcharacterization. To overcome this obstacle, antibodies that do not bindthe postfusion conformation of F and are >10-fold more potent than theprophylactic antibody palivizumab (Synagis®), were identified. Theco-crystal structure for one of these antibodies, D25, in complex withthe F glycoprotein reveals that D25 locks F in its prefusion state.Comparisons of prefusion and postfusion F conformations define therearrangements required to mediate RSV entry. The D25-F glycoproteinstructure reveals a new site-of-vulnerability, antigenic site Ø, at thetop of the F glycoprotein that is prefusion-specific and quaternary incharacter. The prefusion RSV F trimer structure, along with definitionof antigenic site Ø, should enable the design of improved vaccineantigens and guide new approaches for passive prevention of RSV-induceddisease.

Respiratory syncytial virus (RSV) is ubiquitous, infecting nearly allchildren by 3 years of age (Glezen et al., Am. J. Dis. Child., 140, 543(1986)). In the US, RSV bronchiolitis is the leading cause ofhospitalization in infants and a major cause of asthma and wheezingthroughout childhood (Shay et al., JAMA, 282, 1440 (1999); Hall et al.,N. Engl. J. Med., 360, 588 (2009)). Globally, RSV is responsible for66,000-199,000 deaths each year for children younger than five years ofage (Nair et al., Lancet, 375, 1545 (2010)), and accounts for 7% ofdeaths among infants 1 month to 1 year old—more than any other singlepathogen except malaria (Lozano et al., Lancet, 380, 2095 (2013)). Theonly available intervention is passive administration of the licensedmonoclonal antibody palivizumab (Synagis®), which recognizes the RSVfusion (F) glycoprotein (Johnson et al., J. Infect. Dis., 176, 1215(1997); Beeler and van Wyke Coelingh, J. Virol., 63, 2941 (1989)) andreduces incidence of severe disease (The IMpact-RSV Study Group,Pediatrics, 102, 531 (1998)). Clinical evidence that RSV F-specificantibodies can protect against disease has prompted a search for betterantibodies (Collarini et al., J. Immunol., 183, 6338 (2009); Wu et al.,J. Mol. Biol., 368, 652 (2007); Kwakkenbos et al., Nat. Med., 16, 123(2010)) and a concerted effort to develop an effective vaccine (Graham,Immunol. Rev., 239, 149 (2011)).

The RSV F glycoprotein facilitates fusion of viral and cellularmembranes (Walsh and Hruska, J. Virol., 47, 171 (1983)); it is a type Ifusion protein, with a metastable prefusion conformation that storesfolding energy, released during a structural rearrangement to a highlystable postfusion conformation. Three antigenic sites (I, II, and IV)have been found to elicit neutralizing activity (Arbiza et al., J. Gen.Virol., 73, 2225 (1992); Lopez et al., J. Virol., 72, 6922 (1998); Lópezet al., J. Virol., 64, 927 (1990)), and all exist on the postfusion formof F as determined by structural and biophysical studies (McLellan etal., J. Virol., 85, 7788 (2011); Swanson et al., Proc. Natl. Acad. Sci.U.S.A., 108, 9619 (2011)). Absorption of human sera with postfusion F,however, fails to remove the majority of F-specific neutralizingactivity, suggesting that the prefusion form may harbor novelneutralizing antigenic sites (Magro et al., Proc. Natl. Acad. Sci.U.S.A., 109, 3089 (2012)). Despite extensive effort, a homogeneouspreparation of soluble prefusion RSV F has not been obtained. Thus,determination of the prefusion F structure and identification of novelF-specific antigenic sites have become converging priorities fordevelopment of new prophylactic and therapeutic antibodies and vaccines.In line with these objectives, F-specific antibodies that couldneutralize RSV, but not bind to postfusion F were identified, andstructure of RSV F recognized by these antibodies was defined. Theresults reveal the prefusion conformation of RSV F, the mechanism ofneutralization for a category of remarkably potent antibodies, andatomic-level details for a prefusion-specific antigenic site that shouldserve as a target of improved antibody-based therapies and provide abasis for the development of effective vaccine antigens.

Two human antibodies—D25 and AM22—were determined to be ˜50-fold morepotent than palivizumab (FIG. 1A) for neutralizing RSV F, and which alsodo not bind to a soluble form of RSV F stabilized in the postfusionconformation (McLellan et al., J. Virol., 85, 7788 (2011)) (FIG. 1B).D25 and AM22 were previously disclosed (Kwakkenbos et al., Nat. Med.,16, 123 (2010); U.S. Pat. Pub. 2010/0239593; U.S. Pat. Pub.2012/0070446). The lack of D25 and AM22 binding to the postfusion formof RSV F suggested these antibodies might recognize the metastableprefusion conformation.

Structural efforts were focused on the human antibodies, AM22 and D25. A96-well microtiter plate expression format (Pancera et al., PLoS One.2013; 8(2):e55701, 2013, incorporated by reference herein) was used toscreen binding of these antibodies to a panel of RSV F glycoproteinvariants that were captured from cell supernatants on Ni²⁺-NTA ELISAplates. Antibody binding to an F glycoprotein construct (RSV F(+) Fd),comprising RSV F residues 1-513 fused to a C-terminal fibritintrimerization domain was tested (Frank et al., J. Mol. Biol., 308, 1081(2001)). However, complexes were not formed by mixing purified RSV F(+)Fd with purified D25 or AM22 antibody. It was determined thatpurification of the soluble F glycoprotein triggered the metastableprefusion state (Chaiwatpongsakorn et al., J. Virol., 85, 3968 (2011));to overcome this instability, cells expressing RSV F(+) Fd wereincubated with antigen-binding fragments (Fabs) or immunoglobulins (thelatter with an HRV3C protease-cleavage site in the hinge region(McLellan et al., Nature 480, 336, (2011)) in order to trap F in theprefusion state. Alternatively, cells expressing RSV F(+) Fd werecotransfected with separate DNA-expression cassettes encodingantibody-heavy and -light chains (FIG. 5). Optimal expression of a D25-Fglycoprotein complex was obtained from cotransfection of DNA encodingD25 Fab with DNA encoding RSV F(+) Fd; reasonable complex yields werealso observed from the addition of soluble Fab.

Crystallizations were screened for Fab D25 and AM22, alone or in complexwith RSV F(+) Fd. X-ray diffraction data to 1.6 Å resolution wereobtained on hexagonal crystals of Fab D25 by itself, and the structurewas solved by molecular replacement and refined to R_(cryst)/R_(free) of24.5/25.7% (Table 9). Data to 3.6 Å resolution were obtained on cubiccrystals of Fab D25 in complex with RSV F (+) Fd, and this structure wassolved by molecular replacement using the unbound D25 structure andportions of the previously determined postfusion RSV F structure(McLellan et al., J. Virol., 85, 7788 (2011); Swanson et al., Proc.Natl. Acad. Sci. U.S.A., 108, 9619 (2011)) as search models, along withclues from a gold derivative. The structure of the complex was refinedto R_(cryst)/R_(free) of 21.3/26.7% (FIG. 1C) (Table 9).

A complex of one D25 Fab bound to one molecule of the RSV F glycoproteinwas present in the asymmetric unit of the cubic lattice. Three-foldlattice symmetry positioned two other D25-RSV F complexes to generate anextensive RSV F trimeric interface of 2,098 Å². Continuous electrondensity was observed for residues 26 to 513, except for residues 98-136that included the 27 amino-acid fragment removed by proteolytic cleavageof the F₀ precursor to form the F₂ and F₁ subunits (corresponding to N-and C-terminal fragments, respectively) of the mature F glycoprotein.Three sites of N-linked glycosylation were detected in the electrondensity at asparagine residues 27, 70 and 500 (FIG. 2A).

Overall, the D25-bound RSV F structure consists of two lobes packed ateither end of a 7-stranded antiparallel open-ended barrel, two strandsof which (β2 and β7) extend between the two lobes, hydrogen-bonding forover 70 Å and forming integral portions of both lobes and of the centralbarrel. The membrane-proximal lobe, which contains the F₂ N-terminus andF₁ C-terminus, consists of a triple layered β-sandwich and three helices(α8, α9 and α10). Helix α10 forms part of a helix that appeared toextend into the viral membrane and to which the fibrin trimerizationdomain was appended. The membrane-distal lobe, approximately 90 Å fromthe viral membrane, consists of seven helices, packed around athree-stranded antiparallel sheet and a β-hairpin (β3+β4). Extensiveinter-protomer contacts appeared to stabilize the trimeric structure,particularly the hydrophobic N-terminus of the F₁ subunit (also known asthe fusion peptide), which was cradled by the triple β-sandwich from themembrane-proximal lobe of a neighboring protomer. The fusion peptide,contained within the otherwise hollow cavity of the trimer, is connectedto the surface-exposed α2 and α3 helices through a cylindrical openingbetween the protomers that is roughly 10 Å in diameter; this opening maybe used as an exit path for the fusion peptide during triggering.

The structure of the D25-bound F glycoprotein resembled the prefusionstructure of the related parainfluenza virus 5 (PIV5) F glycoprotein(Welch et al., Proc. Natl. Acad. Sci. U.S.A., 109, 16672 (2012); Yin etal., Nature, 439, 38 (2006)) (FIGS. 6 and 7). The D25-bound form of RSVF thus appeared to be in the prefusion conformation (FIG. 2). To definethe structural rearrangements between pre- and post-fusion F, D25-boundform of RSV F was compared with its postfusion conformation, which wasrecently determined (McLellan et al., J. Virol., 85, 7788 (2011);Swanson et al., Proc. Natl. Acad. Sci. U.S.A., 108, 9619 (2011).

Pre- and post-fusion conformations of RSV F revealed dramatic changes inoverall shape, from a relatively compact oval-shaped structure with aheight of 110 Å to an extended cone approximately 50% longer (170 Å)(FIG. 2A). Despite this remarkable change in conformation, the majorityof the F glycoprotein secondary and tertiary structure was preserved inboth pre- and post-fusion states, with 215 residues showing less than 2Å Cα deviation between the two structures (FIGS. 2A,B). Two regions ofstriking conformational change occur. In the membrane-distal lobe, thefusion peptide and five secondary structure elements (α2, α3, β3, β4,and α4) join with the α5-helix to form a single extended postfusionhelix (α5_(post)) of over 100 Å in length, which is capped at itsN-terminus by the fusion peptide (to aid in clarity, secondary structureelements of the postfusion structure are labeled with “post” subscript).In the membrane-proximal lobe, the sole parallel strand (β22) of thetriple β-sandwich—which in the prefusion structure hydrogen bonds toβ1—unravels, allowing the prefusion α10-helix to join with theα5_(post)-helix. Together, the α5_(post) and α10_(post) helicesjuxtapose F₁ N- and C-termini to form the coiled-coil structurecharacteristic of type I fusion proteins in their postfusionconformation (Colman and Lawrence, Nat. Rev. Mol. Cell. Biol., 4, 309(2003)). Overall, portions of the α10 helix move more than 170 Å betweenpre- and post-fusion conformations.

In comparison to the previously reported protease-cleaved, prefusiontype I structures of influenza hemagglutinin (Wilson et al., Nature,289, 366 (1981)), Ebola GP (Lee et al., Nature, 454, 177 (2008)) andPIV5 F (Welch et al., Proc. Natl. Acad. Sci. U.S.A., 109, 16672 (2012)),the location of the RSV fusion peptide is most similar to that ofhemagglutinin (FIG. 7), which is surprising given that PIV5 and RSV areboth paramyxoviruses. The RSV F fusion peptide is buried in the centerof the hollow trimer cavity, and is located more than 40 Å away from thelast visible F₂ residue. This suggests that a substantial structuralrearrangement of the fusion peptide occurs after the F₀ precursor iscleaved by the furin-like host protease to produce F₁/F₂. In addition,dramatic structural rearrangements occur between pre- and post-fusionconformations in both the membrane-proximal and membrane-distal lobes,providing insight into the difficulty of stabilizing the prefusionconformation of RSV F. Unlike PIV5 F and human metapneumovirus F, whichcan be stabilized in the prefusion state solely by appending aGCN4-trimerization motif to the C-terminus (Yin et al., Nature, 439, 38(2006); Wen et al., Nat. Struct. Mol. Biol., 19, 461 (2012)), theprefusion RSV F conformation requires stabilization of both themembrane-proximal lobe (accomplished by appending a fibritintrimerization domain (Frank et al., J. Mol. Biol., 308, 1081 (2001)) andthe membrane-distal lobe (which occurs through binding of the D25antibody).

The D25 antibody recognizes the membrane-distal apex of the RSV Fglycoprotein (FIG. 1C). It binds to a quaternary epitope, with theD25-heavy chain interacting with one protomer (involving 638 Å² ofburied interactive-surface area on RSV) and the D25-light chain bindingto both the same protomer (373 Å²) and a neighboring protomer (112 Å²)(FIG. 3A). RSV F contacts are made by 5 of the 6complementarity-determining loops of D25, with the heavy chain 3^(rd)CDR(CDR H3) interacting with the α4-helix (F₁ residues 196-210) andforming intermolecular hydrogen bonds with F₂ residues 63, 65, 66 and 68in the loop between strand β2 and helix α1. While the secondarystructural elements of the D25 epitope remain mostly unchanged, theirrelative orientation changes substantially, with α4-helix pivoting ˜180°relative to strand β2 in pre- and post-fusion conformations (FIG. 3B).This structural rearrangement explains the failure of D25 to bindpostfusion F molecules and suggests D25 inhibits membrane fusion bystabilizing the prefusion conformation of the trimeric F glycoproteincomplex. Although F proteins from human RSV A and B subtypes are highlyrelated in sequence (447/472 or 94.7% of the amino acids comprising themature F₂/F₁ ectodomain are identical between known subtypes), sixnaturally observed positions of RSV-sequence variation (residues 67 and74 in F₂, and residues 200, 201, 209, and 213 in F₁) are located in theregion bound by D25 (FIG. 3C). Similarly, of the 56 amino acids inbovine RSV F that are not identical to the mature ectodomain of humanRSV F subtype A, 13 are found in this same region (FIG. 3C). Thus, theD25 epitope, at the apex of the prefusion RSV F structure, may be underimmune pressure and serve as a determinant of subtype-specific immunity(Chambers et al., J. Gen. Virol., 73, 1717 (1992)). For example, basedon sequence analysis, a loop region in F glycoproteins was hypothesizedto exist within the Paramyxoviridae family that might be under immunepressure (Chambers et al., J. Gen. Virol., 73, 1717 (1992)). It has beendemonstrated that binding of RSV sub-group specific monoclonalantibodies can be affected by site-directed mutations between F1residues 200 and 216 (Connor et al., J. Med. Virol., 63, 168 (2001)),and that a peptide comprising F1 residues 205-225 could elicitneutralizing activity in rabbits, although a specific epitope was notdefined (Corvaisier et al., Arch. Virol., 142, 1073 (1997)).

To understand the relationship of the D25 epitope relative to epitopesrecognized by other RSV-neutralizing antibodies, competition for D25binding to RSV-infected cells was tested (FIG. 4A). Notably, AM22competed with D25 for RSV F binding, suggesting that they recognized thesame antigenic site. To further define the site recognized by theseantibodies, negative stain EM on Fab-RSV F complexes was performed. EMimages of Fab D25-RSV F complexes resembled the crystal structure of FabD25-RSV F, and also EM images of Fab AM22-RSV F (FIG. 4B). Together,these results suggested antibodies D25 and AM22 recognize the same or ahighly related antigenic site, which was named “antigenic site Ø”.

To characterize antibodies that recognize antigenic site Ø, theirfunctional properties were examined. In addition to their extraordinarypotency and prefusion-specificity (FIG. 1A), all three antibodiesstrongly inhibited fusion when added post-attachment (FIG. 4C), and allthree were unable to block cell-surface attachment (FIG. 4D), suggestingthat the RSV F receptor binds to a region on F not blocked by thesethree antibodies. The receptor-binding domain on the related humanmetapneumovirus F protein is an RGD motif (Cseke et al., Proc. Natl.Acad. Sci. U.S.A., 106, 1566 (2009)) that corresponds to RSV F residues361-363, which reside at the tip of a loop of the central barrel, on theside of the prefusion RSV F trimer not blocked by D25-binding. Althoughthese antibodies do not prevent attachment, the regions of both F₂ andF₁ comprising antigenic site Ø are known to contribute to heparinbinding (Feldman et al., J. Virol., 74, 6442 (2000); Crim et al., J.Virol., 81, 261 (2007)), and it is possible that this region maycontribute to non-specific attachment to heparin sulfate moieties onglycosaminoglycans in concert with the G glycoprotein and other regionsof F. Lastly, AM22 and D25 antibodies neutralized similarly in both Faband immunoglobulin contexts (FIG. 8), indicating that avidity did notplay a dominant role as it does for some influenza-virus antibodies(Ekiert et al., Nature, 489, 526 (2012)). Overall, the sharedbinding-specificity and neutralization phenotypes of D25 and AM22 andsuggest that these properties may be characteristic of antibodies thatrecognize antigenic site Ø. By contrast, none of the antibodies thatrecognize other antigenic sites on RSV F associated with neutralizingactivity (sites I, II, and IV) share similar properties of neutralizingpotency and prefusion F specificity (FIGS. 9A-9B).

Despite antigenic site Ø being partially shielded from immunerecognition by multiple mechanisms including conformational masking (itis only present in the metastable prefusion state), quaternary assembly(the site is shared by RSV protomers), antigenic variation (it is one ofthe most variable portions of RSV F), and glycan shielding (the N-linkedglycan attached to Asn70 is at the top of the prefusion F trimer), allthree prefusion-specific antibodies appear to target a similar epitope.The location of antigenic site Ø at the apex of the prefusion F trimershould be readily accessible even on the crowded virion surface, whichmay explain the observation that most neutralizing activity in humansera induced by natural RSV infection is directed against the prefusionform of RSV F (Magro et al., Proc. Natl. Acad. Sci. U.S.A., 109, 3089(2012), although other prefusion-specific antigenic sites cannot beruled out. The high potency of antibodies against antigenic site Øsuggests they could be developed for passive prophylaxis of RSV-induceddisease in neonates. Also, vaccine-based prefusion specific antibodyelicitation may be assisted by stabilization of the prefusion form ofRSV F, perhaps facilitated by linking mobile and immobile portions ofthe F structure through structure-based design of RSV F variants withdisulfide bonds. It is noted that prefusion-stabilized F contains all ofthe previously characterized neutralizing epitopes as well as antigenicsite Ø. Definition of the D25-RSV F structure thus provides the basisfor multiple new approaches to prevent RSV-induced disease.

Materials and Methods

Viruses and Cells.

Viral stocks were prepared and maintained as previously described(Graham et al., J. Med. Virol., 26, 153 (1988)) RSV-expressing GreenFluorescent Protein (GFP) RSV-GFP was constructed as previously reported(Hallak et al., Virology. 271, 264 (2000)). The titer of the RSV-GFPstocks used for flow cytometry-based neutralization and fusion assayswas 2.5×10⁷ pfu/ml. The titer of the RSV A2 stock used for attachmentassay was 1.02×10⁸ pfu/ml. HEp-2 cells were maintained in Eagle'sminimal essential medium containing 10% fetal bovine serum (10% EMEM)and were supplemented with glutamine, penicillin and streptomycin.

Creation of Antibody Expression Plasmids.

DNA encoding antibody heavy and light variable regions werecodon-optimized for human expression and synthesized. AM22 and D25 heavyand light variable regions were subcloned into pVRC8400 expressionplasmids containing in-frame human constant domains (IgG1 for heavychain and kappa for light chain). Variants of the AM22 and D25 heavychain expression plasmids were made by inserting either an HRV3Cprotease site (GLEVLFQGP; SEQ ID NO: 355) or a stop codon into the hingeregion.

Expression and Purification of Antibodies and Fab Fragments.

Antibodies were expressed by transient co-transfection of heavy andlight chain plasmids into HEK293F cells in suspension at 37° C. for 4-5days. The cell supernatants were passed over Protein A agarose, andbound antibodies were washed with PBS and eluted with IgG elution bufferinto 1/10th volume of 1 M Tris-HCl pH 8.0. AM22 and D25 Fabs werecreated by digesting the IgG with Lys-C. The digestion was inhibited bythe addition of Complete protease inhibitor cocktail tablets, and theFab and Fc mixtures was passed back over Protein A agarose to remove Fcfragments. The Fab that flowed through the column was further purifiedby size exclusion chromatography.

RSV Neutralization Assays.

Antibody-mediated neutralization was measured by a flow cytometryneutralization assay (Chen et al., J. Immunol. Methods, 362, 180 (2010).Briefly, HEp-2 cells were infected with RSV-GFP and infection wasmonitored as a function of GFP expression at 18 hours post-infection byflow cytometry. Data were analyzed by curve fitting and non-linearregression (GraphPad Prism, GraphPad Software Inc., San Diego Calif.).

Postfusion RSV F-Binding Assay.

Purified, soluble RSV F protein in the postfusion conformation wasprepared as described in (McLellan et al., J. Virol., 85, 7788 (2011). Akinetic ELISA was used to test binding of monoclonal antibodies topostfusion RSV F as described previously (McLellan et al., J. Mol.Biol., 409, 853 (2011). Briefly, 96-well Ni²⁺-NTA-coated plates(ThermoFisher Scientific) were coated with 100 μl postfusion RSV F (1μg/ml) for one hour at room temperature. 100 μl of diluted antibody wasadded to each well and incubated for one hour at room temperature. Boundantibodies were detected by incubating the plates with 100 μlHRP-conjugated goat anti-mouse IgG antibody (Jackson ImmunoResearchLaboratories, West Grove, Pa.) or HRP-conjugated anti-human IgG (SantaCruz Biotechnology, Inc, Santa Cruz, Calif.) for 1 hour at roomtemperature. Then, 100 μl of Super AquaBlue ELISA substrate(eBioscience, San Diego Calif.) was added to each well and plates wereread immediately using a Dynex Technologies microplate reader at 405 nm(Chantilly, Va.). Between steps, plates were washed with PBS-T.

Crystallization and X-Ray Data Collection of Unbound D25 Fab.

Crystallization conditions were screened using a Cartesian Honeybeecrystallization robot, and initial crystals were grown by the vapordiffusion method in sitting drops at 20° C. by mixing 0.2 μl of D25 Fabwith 0.2 μl of reservoir solution (22% (w/v) PEG 4000, 0.1 M sodiumacetate pH 4.6). Crystals were manually reproduced in hanging drops bycombining protein and reservoir solution at a 2:1 ratio. Crystals wereflash frozen in liquid nitrogen in 27.5% (w/v) PEG 4000, 0.1 M sodiumacetate pH 4.5, and 15% (v/v) 2R,3R-butanediol. X-ray diffraction datato 1.6 Å were collected at a wavelength of 1.00 Å at the SER-CATbeamline ID-22 (Advanced Photon Source, Argonne National Laboratory).

Structure Determination and Refinement of Unbound D25 Fab.

X-ray diffraction data were integrated and scaled with the HKL2000 suite(Otwinowski and Minor, in Methods Enzymol. (Academic Press, vol. 276,pp. 307-326, 1997)), and a molecular replacement solution using Igdomains from PDB ID: 3 GBM (Ekiert et al., Science, 324, 246 (2009)) and3IDX (Chen et al., Science, 326, 1123 (2009)) as search models wasobtained using PHASER (McCoy et al., J. Appl. Crystallogr., 40, 658(2007)). Manual model building was carried out using COOT (Emsley etal., Acta Crystallogr D Biol Crystallogr, 66, 486 (2010)), andrefinement of individual sites, TLS parameters, and individual B-factorswas performed in PHENIX (Adams et al., Acta Crystallogr D BiolCrystallogr, 66, 213 (2010)). The electron density for the D25 variabledomains was excellent, but the electron density for the constant domainswas poor, possibly a result of flexibility in the elbow angle. Finaldata collection and refinement statistics are presented in Table 8.

Expression and Purification of RSV F(+) Fd in Complex with D25 Fab.

The RSV F (+) Fd protein construct was derived from the A2 strain(accession P03420) with three naturally occurring substitutions (P102 Å,I379V, and M447V) to enhance expression. A mammalian codon-optimizedgene encoding RSV F residues 1-513 with a C-terminal T4 fibritintrimerization motif (Frank et al., J. Mol. Biol., 308, 1081 (2001)),thrombin site, 6×His-tag, and StreptagII was synthesized and subclonedinto a mammalian expression vector derived from pLEXm (Aricescu et al.,Acta Crystallogr D Biol Crystallogr, 62, 1243 (2006)). Plasmidsexpressing RSV F(+) Fd, the D25 light chain, and the D25 heavy chain(with or without a stop codon in the hinge region) were simultaneouslytransfected into HEK293 GnTI^(−/−) cells (Reeves et al., Proc. Natl.Acad. Sci. U.S.A., 99, 13419 (2002)) in suspension. Alternatively, justthe RSV F(+) Fd plasmid could be transfected, with purified D25 Fabadded to the GnTI^(−/−) cells 3 hours post-transfection. After 4-5 days,the cell supernatant was harvested, centrifuged, filtered andconcentrated. The complex was initially purified via Ni²⁺-NTA resin(Qiagen, Valencia, Calif.) using an elution buffer consisting of 20 mMTris-HCl pH 7.5, 200 mM NaCl, and 250 mM imidazole pH 8.0. The complexwas then concentrated and further purified over StrepTactin resin as perthe manufacturer's instructions (Novagen, Darmstadt, Germany). After anovernight incubation with thrombin protease (Novagen) to remove the Hisand Strep tags, an excess of D25 Fab was added to the complex, which wasthen purified on a Superose6 gel filtration column (GE Healthcare) witha running buffer of 2 mM Tris-HCl pH 7.5, 350 mM NaCl, and 0.02% NaN₃.The eluted complex was diluted with an equal volume of water andconcentrated to ˜5 mg/ml. Similar procedures were used to express andpurify AM22 Fab complexes.

Crystallization and X-Ray Data Collection of RSV F(+) Fd in Complex withD25 Fab.

Initial crystals were grown by the vapor diffusion method in sittingdrops at 20° C. by mixing 0.1 μl of RSV F(+) Fd bound to D25 Fab with0.1 μl of reservoir solution (40% (w/v) PEG 400, 5% (w/v) PEG 3350, and0.1 M sodium acetate pH 5.5) (Majeed et al., Structure, 11, 1061(2003)). Crystals were manually reproduced in hanging drops, and thecrystal that diffracted to 3.6 Å was grown using a reservoir solutioncontaining 30% (w/v) PEG 400, 3.75% (w/v) PEG 3350, 0.1 M HEPES pH 7.5,and 1% (v/v) 1,2-butanediol. The crystal was directly transferred fromthe drop into the cryostream, and X-ray diffraction data were collectedremotely at a wavelength of 1.00 Å at the SER-CAT beamline ID-22.

Structure Determination and Refinement of RSV F(+)Fd in Complex with D25Fab.

X-ray diffraction data were integrated and scaled with the HKL2000 suite(Otwinowski and Minor, in Methods Enzymol. (Academic Press, vol. 276,pp. 307-326, 1997)), and a molecular replacement solution was obtainedby PHASER (McCoy et al., J. Appl. Crystallogr., 40, 658 (2007)) usingthe unbound D25 Fab structure and residues 29-42, 49-60, 78-98, 219-306,313-322, 333-343, and 376-459 from the postfusion RSV F structure (PDBID: 3RRR, McLellan et al., J. Virol., 85, 7788 (2011)) as search models.Six sites from a NaAuCl4 derivative mapped to known reactive side chains(F residues Met97/His159, Met264/Met274, His317, and Met396; D25 heavychain residues Met19/His82 and His 59). Manual model building wascarried out using COOT (Emsley et al., Acta Crystallogr D BiolCrystallogr, 66, 486 (2010)), with secondary structure elements beingbuilt first. Refinement of individual sites, TLS parameters, andindividual B-factors was performed in PHENIX (Adams et al., ActaCrystallogr D Biol Crystallogr, 66, 213 (2010)), using the unbound D25Fab structure, and portions of the postfusion RSV F structure asreference models during the refinement. All RSV F residues in the matureprotein were built except for those residues in F₂ C-terminal to Met97.Final data collection and refinement statistics are presented in Table9.

RSV F Competition Binding Assay.

Competition binding of antibodies was performed on RSV infected HEp-2cells. HEp-2 cells were infected with 3 MOI (multiplicity of infection)of RSV for 18-20 hours. After infection, cells were separated using celldissociation solution (Cellstripper, Mediatech Inc., Herndon, Va.), andwashed with PBS. Cells were seeded at 5×10⁴/well in 96-well U-bottomplates in PBS. Monoclonal antibodies AM22, D25, and 101F were dilutedstarting at a concentration of 100 μg/ml, and added to HEp-2 cells.After 30 minutes 100 ul of Alexa 488 conjugated D25 was added at aconcentration of 1 μg/ml and incubated at 4° C. for one hour. Cells werewashed once with PBS, and then fixed with 0.5% paraformaldehyde. Thebinding of D25-Alexa 488 on cells was measured by flow cytometry (LSR IIinstrument, Becton Dickinson, San Jose, Calif.). Data were analyzed byusing FlowJo software, version 8.5 (Tree Star, San Carlos, Calif.).

Negative Staining Electron Microscopy Analysis.

Samples were adsorbed to freshly glow-discharged carbon-coated grids,rinsed shortly with water, and stained with freshly made 0.75% uranylformate. Images were recorded on an FEI T20 microscope with an Eagle CCDcamera. Image analysis and 2D averaging was performed with Bsoft(Heymann and Belnap, J. Struct. Biol., 157, 3 (2007) and EMAN (Ludtke etal., J. Struct. Biol., 128, 82 (1999)).

RSV Virus-to-Cell Fusion Inhibition Assay.

The ability of antibodies to inhibit RSV virus-to-cell fusion wasmeasured as described previously (McLellan et al., J. Virol., 84, 12236(2010)). Briefly, HEp-2 cells were seeded in 96-well plates, culturedfor 24 hours at 37° C., and then chilled at 4° C. for one hour prior toassay. RSV-GFP was added to pre-chilled cells at 4° C., and then cellswere washed in cold PBS to remove unbound virus. Serially-dilutedantibodies were added to chilled cells and incubated for 1 hour at 4°C., before transferring to 37° C. for 18 hours. After incubation, cellswere trypsinized, fixed in 0.5% paraformaldehyde, and analyzed by flowcytometry to determine the frequency of GFP-expressing cells.

RSV Attachment Inhibition Assay.

The ability of antibodies to inhibit RSV attachment to cells wasmeasured as described previously (McLellan et al., J. Virol., 84, 12236(2010)). Briefly, HEp-2 cells were dispersed into media, washed withcold PBS, seeded in 96-well v-bottom plates, and chilled for 1 hour at4° C. before use. Antibodies and heparin, a known RSV attachmentinhibitor, were distributed in serial dilutions, then mixed with RSV A2strain virus for one hour at 37° C. Medium from chilled cells wasremoved after centrifugation and virus or mixtures of virus and reagentswere added to chilled cells and incubated for 1 hour at 4° C. Afterincubation, cells were washed in cold PBS to remove unbound virus, andfixed with 0.5% paraformaldehyde. Viruses bound on cells were detectedwith FITC-conjugated goat anti-RSV antibody. Cells were washed in coldPBS and evaluated by flow cytometry. Median fluorescence intensities ofbound virus were analyzed with FlowJo software, version 8.5 (Tree Star,San Carlos, Calif.).

TABLE 9 Crystallographic data collection and refinement statistics. D25Fab D25 Fab + RSV F Data collection Space group P6₁22 P2₁3 Cellconstants a, b, c (Å) 108.7, 108.7, 139.9 152.3, 152.3, 152.3 α, β, γ(°) 90.0, 90.0, 120.0 90.0, 90.0, 90.0 Wavelength (Å) 1.00 1.00Resolution (Å) 50.0-1.6 (1.63-1.60) 50.0-3.6 (3.73-3.60) R_(merge) 11.2(68.0) 12.7 (81.4) I/σI 27.3 (2.1) 16.4 (2.0) Completeness (%) 98.3(86.1) 99.6 (99.3) Redundancy 11.0 (5.3) 6.5 (5.2) Refinement Resolution(Å) 35.4-1.6 (1.62-1.60) 42.2-3.6 (3.88-3.60) Unique reflections 63,360(2,241) 13,877 (2,742) R_(work)/R_(free) (%) 24.1/25.5 21.3/26.7 No.atoms Protein 3,305 6,778 Ligand/ion 0 0 Water 270 0 B-factors (Å²)Protein 53.0 128.1 Ligand/ion — — Water 44.1 — R.m.s. deviations Bondlengths (Å) 0.007 0.003 Bond angles (°) 1.20 0.91 Ramachandran Favored(%) 96.5 92.0 Allowed (%) 3.0 7.3 Outliers (%) 0.5 0.7

Example 2 Stabilization of RSV F Proteins

This example illustrates design of exemplary RSV F proteins stabilizedin a prefusion conformation. The crystal structure of the RSV F proteinin complex with D25 Fab (i.e., in a prefusion conformation) compared tothe structure of the postfusion RSV F protein (disclosed, e.g., inMcLellan et al., J. Virol., 85, 7788, 2011, with coordinates depositedas PDB Accession No. 3RRR) shows dramatic structural rearrangementsbetween pre- and post-fusion conformations in both the membrane-proximaland membrane-distal lobes, providing guidance for the stabilization ofthe prefusion conformation of RSV F. Based on a comparison of the pre-and post-fusion RSV F structures, there are two regions that undergolarge conformational changes, located at the N- and C-termini of the F₁subunit. For example, as illustrated in FIG. 2, the positions 137-216and 461-513 of the F₁ polypeptide undergo structural rearrangementbetween the Pre- and Post-F protein conformations, whereas positions271-460 of the F₁ polypeptide remain relatively unchanged. This exampleillustrates several strategies of stabilizing the RSV F protein in itsprefusion conformation.

To stabilize the N-terminal region of F₁, which is a component ofantigenic site Ø and is involved in binding to antibody D25, variousstrategies have been designed, including introduction of intra-protomerdisulfide bonds, inter-protomer disulfide bonds, cavity filling aminoacid substitutions, repacking substitutions, introduction of N-linkedglycosylation sites, and combinations thereof.

Intra-Protomer Disulfide Bonds.

Introduction of two cysteine residues that are within a sufficientlyclose distance to form an intra-protomer disulfide bond in theprefusion, but not postfusion, conformation can lock the F protein inthe prefusion conformation. An intra-molecular disulfide bond can beformed within a single F₂/F₁ protomer within the trimer, and thus wouldnot cross-link the three protomers together. Specifically, a disulfidebond formed between a region that changes conformation and a region thatdoes not change conformation in the pre- and post-fusion structuresshould lock the protein in the prefusion conformation. One example isthat of the S155C/S290C mutant, where Ser155 is located in a region thatchanges conformation, whereas Ser290 is in a region that does not changeconformation. Additionally, formation of a disulfide bond between tworegions that both change conformation, such as two residues locatedwithin F₁ positions 137-216, or two residues located within F₁ positions461-513, or one residue within F₁ positions 137-216 and the secondwithin F₁ positions 461-513, may also be sufficient to lock the proteinin the prefusion conformation.

Using the methods described above, several pairs of residues of the RSVF protein were determined to be in close enough proximity in theprefusion conformation, but not the postfusion conformation, to form anintra-protomer disulfide bond if cysteines were introduces at thecorresponding residue pair positions. These residue pairs, as well asthe corresponding amino acid substitutions needed to introduce cysteineresidues at these positions, are indicated in Table 10, below. Table 10also lists a SEQ ID NO containing the indicated substitutions, andcorresponding to a precursor F₀ construct including a signal peptide, F₂polypeptide (positions 26-109), pep27 polypeptide (positions 27-136), F₁polypeptide (positions 137-513), a trimerization domain (a Foldondomain) and a thrombin cleavage site (LVPRGS) and purification tags(his-tag (HHHHHH) and Strep Tag II (SAWSHPQFEK)).

TABLE 10 Exemplary Cross-Linked Cysteine Pairs for Intra-ProtomerDisulfide Bond Stabilization F protein Residue A.A. substitutionsPair(s) for corresponding to Cysteine Substitution SEQ ID NO: 1 SEQ IDNO F₁ Substitutions 155 and 290 S155C and S290C 185 151 and 288 G151Cand I288C 189 137 and 337 F137C and T337C 213 397 and 487 T397C andE487C 247 138 and 353 L138C and P353C 257 341 and 352 W341C and F352C267 403 and 420 S403C and T420C 268 319 and 413 S319C and I413C 269 401and 417 D401C and Y417C 270 381 and 388 L381C and N388C 271 320 and 415P320C and S415C 272 319 and 415 S319C and S415C 273 331 and 401 N331Cand D401C 274 320 and 335 P320C and T335C 275 406 and 413 V406C andI413C 277 381 and 391 L381C and Y391C 278 357 and 371 T357C and N371C279 403 and 417 S403C and Y417C 280 321 and 334 L321C and L334C 281 338and 394 D338C and K394C 282 288 and 300 I288C and V300C 284 F₂ and F₁Substitutions 60 and 194 E60C and D194C 190 33 and 469 Y33C and V469C211 54 and 154 T54C and V154C 212 59 and 192 I59C and V192C 246 46 and311 S46C and T311C 276 48 and 308 L48C and V308C 283 30 and 410 E30C andL410C 285

Intermolecular Disulfide Bonds.

Introduction of two cysteine residues that are within a sufficientlyclose distance to form an inter-protomer disulfide bond in theprefusion, but not postfusion, conformation can lock the F protein inthe prefusion conformation. An inter-protomer disulfide bond would beformed between adjacent protomers within the trimer, and thus wouldcross-link the three protomers together. Specifically, a disulfide bondformed between a region that changes conformation and a region that doesnot change conformation in the pre- and post-fusion structures shouldlock the protein in the prefusion conformation. One example is that ofthe A 153C/K461C mutant, where Ala153 is located in a region thatchanges conformation, whereas Lys461 is in a region that does not changeconformation. Additionally, formation of a disulfide bond between tworegions that both change conformation, such as two residues locatedwithin F₁ positions 137-216, or two residues located within F₁ positions461-513, or one residue within F₁ positions 137-216 and the secondwithin F₁ positions 461-513, may also be sufficient to lock the proteinin the prefusion conformation.

Using the methods described above, several pairs of residues of the RSVF protein were determined to be in close enough proximity in theprefusion conformation, but not the post-fusion conformation, to form aninter-protomer disulfide bond if cysteines were introduced at thecorresponding residue pair positions. These residue pairs, as well asthe corresponding amino acid substitutions needed to introduce cysteineresidues at these positions, are indicated in Table 11, below. Table 11also lists a SEQ ID NO containing the indicated substitutions, andcorresponding to a precursor F₀ construct also including a signalpeptide, F₂ polypeptide (positions 26-109), pep27 polypeptide (positions27-136), F₁ polypeptide (positions 137-513), a trimerization domain (aFoldon domain) and a thrombin cleavage site (LVPRGS) and purificationtags (his-tag (HHHHHH) and Strep Tag II (SAWSHPQFEK)).

TABLE 11 Exemplary Cross-Linked Cysteine Pairs for Inter-ProtomerDisulfide Bond Stabilization F protein A.A. substitutions Residuecorresponding pair(s) to SEQ ID NO: 1 SEQ ID NO F₁ Substitutions 400 and489 T400C and D489C 201 144 and 406 V144C and V406C 202 153 and 461A153C and K461C 205 149 and 458 A149C and Y458C 207 143 and 404 G143Cand S404S 209 346 and 454 S346C and N454C 244 399 and 494 K399C andQ494C 245 146 and 407 S146C and I407C 264 374 and 454 T374C and N454C265 369 and 455 T369C and T455C 266 402 and 141 V402C and L141C 302 F₂and F₁ Substitutions 74 and 218 A74C and E218C 243

Additionally, multiple stabilizing mutations described herein can becombined to generate a PreF antigen containing more than one stabilizingmutation. Examples of such constructs containing a first and secondresidue pair that form an intra- or an inter-protomer disulfide bond areprovided in Table 12, below. Table 12 also lists a SEQ ID NO containingthe indicated substitutions, and corresponding to a precursor F0construct also including a signal peptide, F₂ polypeptide (positions26-109), pep27 polypeptide (positions 27-136), F₁ polypeptide (positions137-513), a trimerization domain (a Foldon domain) and a thrombincleavage site (LVPRGS) and purification tags (his-tag (HHHHHH) and StrepTag II (SAWSHPQFEK)).

TABLE 12 Exemplary Cross-Linked Cysteine Pairs for Combinations ofIntra- and Inter-Protomer Disulfide Bond Stabilization. F proteinResidue pair(s) Substitutions SEQ ID NO 155 and 290 (Intra); S155C andS290C; 303 and 402 and 141 (Inter) and V402C and L141C 155 and290(Intra); S155C and S290C; 263 and 74 and 218 and A74C and E218C

Further, amino acids can be inserted (or deleted) from the F proteinsequence to adjust the alignment of residues in the F protein structure,such that particular residue pairs are within a sufficiently closedistance to form an intra- or inter-protomer disulfide bond in theprefusion, but not postfusion, conformation, which, as discussed above,will stabilize the F protein in the prefusion conformation. Examples ofsuch modification are provided in Table 13, below. Table 13 also lists aSEQ ID NO containing the indicated substitutions, and corresponding to aprecursor F₀ construct also including a signal peptide, F₂ polypeptide(positions 26-109), pep27 polypeptide (positions 27-136), F₁ polypeptide(positions 137-513), a trimerization domain (a Foldon domain) and athrombin cleavage site (LVPRGS) and purification tags (his-tag (HHHHHH)and Strep Tag II (SAWSHPQFEK)).

TABLE 13 Using amino acid insertions to orient F proteins to acceptinter-intra-protomer disulfide bonds, or combinations thereof. SEQ ID Fprotein Residue pair(s) Substitutions NO 155 and 290 (Intra); and 146and 460 (Inter); S155C and S290C; and S146C and N460C; 258 G insertionbetween position 460/461 G insertion between position 460/461 155 and290 (Intra); and 345 and 454(Inter); S155C and S290C; and N345C andN454G; 259 C insertion between positions 453/454 C insertion betweenpositions 453/454 155 and 290 (Intra); and 374 and 454(Inter); S155C andS290C; and T374C and N454G; 260 C insertion between positions 453/454 Cinsertion between positions 453/454 155 and 290 (Intra); and 239 and279(Inter); S155C and S290C; and S238G and Q279C; 261 C insertionbetween positions 238/239 C insertion between positions 238/239 155 and290 (Intra); and 493 paired with C S155C and S290C; and S493C pairedwith a 262 insertion between positions 329/330 C insertion betweenpositions 329/330 183 and 428 (Inter), G insertion between N183C andN428C; G insertion between 296 positions 182/183 positions 182/183 183and 428 (Inter), C insertion between N183C and N427G; C insertionbetween 297 positions 427/428 positions 427/428 155 and 290 (Intra); and183 and 428(Inter); S155C and S290C; and N183C and N428C; 298 Ginsertion between positions 182/183 G insertion between positions182/183 155 and 290 (Intra); and 183 and 428(Inter); S155C and S290C;and N183C and N427G; 299 C insertion between positions 427/428 Cinsertion between positions 427/428 145 and 460 (Inter), AA insertionbetween S145C and 460C; AA insertion between 338 positions 146/147positions 146/147 183 and 423 (Inter), AAA insertion between N183C andK423C; AAA insertion between 339 positions 182/183 positions 182/183 330and 430 (Inter); CAA insertion between A329C and S430C; and a CAAinsertion 340 positions 329/330 between positions 329/330

Cavity-Filling Substitutions.

Comparison of the crystal structure of the RSV F protein in complex withD25 Fab (i.e., in a prefusion conformation) compared to the structure ofthe postfusion RSV F protein (disclosed, e.g., in McLellan et al., J.Virol., 85, 7788, 2011; structural coordinates of the RSV F protein inits postfusion conformation are deposited in the Protein Data Bank (PDB)as PDB Accession No. 3RRR) identifies several internal cavities orpockets in the prefusion conformation that must collapse for F totransition to the postfusion conformation. These cavities are listed inTable 14, below. Accordingly, filling these internal cavities stabilizesF in the prefusion state, by preventing transition to the postfusionconformation. Cavities are filled by substituting amino acids with largeside chains for those with small sidechains. The cavities can beintra-protomer cavities, or inter-protomer cavities. One example of aRSV F cavity-filling modification to stabilize the RSV protein in itsprefusion conformation is the S190F/V207L mutant.

Using this strategy, several cavity filling modifications wereidentified to stabilize the RSV F protein in its prefusion conformation.These modifications, are indicated in Table 14, below. Table 14 alsolists a SEQ ID NO containing the indicated substitutions, andcorresponding to a precursor F₀ construct including a signal peptide, F₂polypeptide (positions 26-109), pep27 polypeptide (positions 27-136), F₁polypeptide (positions 137-513), a trimerization domain (a Foldondomain) and a thrombin cleavage site (LVPRGS) and purification tags(his-tag (HHHHHH) and Strep Tag II (SAWSHPQFEK)).

TABLE 14 Exemplarity cavity-filling amino acid substitution Cavity A.A.Substitutions SEQ ID NO: Ser190 190F and 207L 191 Val207 207L and 220L193 Ser190 and Val296 296F and 190F 196 Ala153 and Val207 220L and 153W197 Val207 203W 248 Ser190 and Val207 83W and 260W 192 Val296 58W and298L 195 Val90 87F and 90L 194

The indicated cavities are referred to by a small residue abutting thecavity that can be mutated to a larger residue to fill the cavity. Itwill be understood that other residues (besides the one the cavity isnamed after) could also be mutated to fill the same cavity.

Repacking Substitutions.

Additionally, the prefusion conformation of the RSV F protein may bestabilized by increasing the interactions of neighboring residues, suchas by enhancing hydrophobic interactions or hydrogen-bond formation.Further, the prefusion conformation of the RSV F protein may bestabilized by reducing unfavorable or repulsive interactions ofneighboring residues that lead to metastability of the prefusionconformation. This can be accomplished by eliminating clusters ofsimilarly charged residues. Examples of such modifications are indicatedin Table 15, below. Table 15 also lists a SEQ ID NO containing theindicated substitutions, and corresponding to a precursor F₀ constructincluding a signal peptide, F₂ polypeptide (positions 26-109), pep27polypeptide (positions 27-136), F₁ polypeptide (positions 137-513), atrimerization domain (a Foldon domain) and a thrombin cleavage site(LVPRGS) and purification tags (his-tag (HHHHHH) and Strep Tag II(SAWSHPQFEK)).

TABLE 15 Repacking Amino Acid Substitutions SEQ ID Substitutions NOI64L, I79V, Y86W, L193V, L195F, Y198F, I199F, 227 L203F, V207L, I214LI64L, I79L, Y86W, L193V, L195F, Y198F, I199F, 228 L203F, I214L I64W,I79V, Y86W, L193V, L195F, Y198F, I199F, 229 L203F, V207L, I214L I79V,Y86F, L193V, L195F, Y198F, I199F, L203F, 230 V207L, I214L I64V, I79V,Y86W, L193V, L195F, Y198F, I199Y, 231 L203F, V207L, I214L I64F, I79V,Y86W, L193V, L195F, Y198F, I199F, 232 L203F, V207L, I214L I64L, I79V,Y86W, L193V, L195F, I199F, L203F, 233 V207L, I214L V56I, T58I, V164I,L171I, V179L, L181F, V187I, 234 I291V, V296I, A298I V56I, T58I, V164I,V179L, T189F, I291V, V296I, 235 A298I V56L, T58I, L158W, V164L, I167V,L171I, V179L, 236 L181F, V187I, I291V, V296L V56L, T58I, L158Y, V164L,I167V, V187I, T189F, 237 I291V, V296L V56I, T58W, V164I, I167F, L171I,V179L, L181V, 238 V187I, I291V, V296I V56I, T58I, I64L, I79V, Y86W,V164I, V179L, T189F, 239 L193V, L195F, Y198F, I199F, L203F, V207L,I214L, I291V, V296I, A298I V56I, T58I, I79V, Y86F, V164I, V179L, T189F,L193V, 240 L195F, Y198F, I199F, L203F, V207L, I214L, I291V, V296I, A298IV56I, T58W, I64L, I79V, Y86W, V164I, I167F, L171I, 241 V179L, L181V,V187I, L193V, L195F, Y198F, I199F, L203F, V207L, I214L, I291V, V296IV56I, T58W, I79V, Y86F, V164I, I167F, L171I, V179L, 242 L181V, V187I,L193V, L195F, Y198F, I199F, L203F, V207L, I214L, I291V, V296I D486N,E487Q, D489N, and S491A 249 D486H, E487Q, and D489H 250 T400V, D486L,E487L, and D489L 251 T400V, D486I, E487L, and D489I, 252 T400V, S485I,D486L, E487L, D489L, Q494L, and K498L 253 T400V, S485I, D486I, E487L,D489I, Q494L, and K498L 254 K399I, T400V, S485I, D486L, E487L, D489L,Q494L, 255 E497L, and K498L K399I, T400V, S485I, D486I, E487L, D489I,Q494L, 256 E497L, and K498L L375W, Y391F, and K394M 286 L375W, Y391F,and K394W 287 L375W, Y391F, K394M, D486N, E487Q, D489N, and 288 S491AL375W, Y391F, K394M, D486H, E487Q, and D489H 289 L375W, Y391F, K394W,D486N, E487Q, D489N, and 290 S491A L375W, Y391F, K394W, D486H, E487Q,and D489H 291 L375W, Y391F, K394M, T400V, D486L, E487L, D489L, 292Q494L, and K498M L375W, Y391F, K394M, T400V, D486I, E487L, D489I, 293Q494L, and K498M L375W, Y391F, K394W, T400V, D486L, E487L, D489L, 294Q494L, and K498M L375W, Y391F, K394W, T400V, D486I, E487L, D489I, 295Q494L, and K498M F137W and R339M 326 F137W and F140W 327 F137W, F140W,and F488W 328 D486N, E487Q, D489N, S491A, and F488W 329 D486H, E487Q,D489H, and F488W 330 T400V, D486L, E487L, D489L, and F488W 331 T400V,D486I, E487L, D489I, and F488W 332 D486N, E487Q, D489N, S491A, F137W,and F140W 333 D486H, E487Q, D489H, F137W, and F140W 334 T400V, D486L,E487L, D489L, F137W, and F140W 335 L375W, Y391F, K394M, F137W, and F140Wor 336 L375W, Y391F, K394M, F137W, F140W, and R339M 337

Glycosylation Mutations.

Additionally, introduction of N-linked glycosylation sites that would besolvent-accessible in the prefusion RSV F conformation butsolvent-inaccessible in the postfusion RSV F conformation may stabilizeRSV F in the prefusion state by preventing adoption of the postfusionstate. To create an N-linked glycosylation site, the sequenceAsn-X-Ser/Thr (where X is any amino acid except Pro) may be introduced.This can be accomplished by substitution of a Ser/Thr amino acid tworesidues C-terminal to a native Asn residue, or by substitution of anAsn amino acid two residues N-terminal to a native Ser/Thr residue, orby substitution of both an Asn and Ser/Thr residue separated by onenon-proline amino acid.

Using this strategy, several locations for N-linked glycosylation sitesthat would be solvent-accessible in the prefusion RSV F conformation butsolvent-inaccessible in the postfusion RSV F conformation wereidentified. These modifications are indicated in Table 16, below. Table16 also lists the SEQ ID NO containing the indicated substitutions, andcorresponding to a precursor F₀ construct including a signal peptide, F₂polypeptide (positions 26-109), pep27 polypeptide (positions 27-136), F₁polypeptide (positions 137-513), a trimerization domain (a Foldondomain) and a thrombin cleavage site (LVPRGS) and purification tags(his-tag (HHHHHH) and Strep Tag II (SAWSHPQFEK)).

TABLE 16 Exemplary N-linked glycosylation N-linked glycosylationExemplary Exemplary position substitutions SEQ ID NO 506 I506N and K508T198 175 A177S 199 178 V178N 200 276 V278T 203 476 Y478T 204 185 V185Nand V187T 214 160 L160N and G162S 215 503 L503N and a F505S 216 157V157N 217

Example 3 Stabilizing the Membrane Proximal Lobe of PreF Antigens

As discussed above, the crystal structure of the RSV F protein incomplex with D25 Fab (i.e., in a prefusion conformation) compared to thestructure of the postfusion RSV F protein ((disclosed, e.g., in McLellanet al., J. Virol., 85, 7788, 2011, with coordinates deposited as PDBAccession No. 3RRR)) shows dramatic structural rearrangements betweenpre- and post-fusion conformations in the membrane-distal lobe. Based ona comparison of the pre- and post-fusion RSV F structures, there are tworegions that undergo large conformational changes, located at the N- andC-termini of the F₁ subunit. For example, as illustrated in FIG. 2, thepositions 137-216 and 461-513 of the F₁ polypeptide undergo structuralrearrangement between the Pre- and Post-F protein conformations, whereaspositions 271-460 of the F₁ polypeptide remain relatively unchanged.This example illustrates several strategies of stabilizing theC-terminal region of F₁, which includes the membrane proximal lobe ofthe RSV F protein. Various strategies have been identified, includingintroduction of a trimerization domain (as discussed above),introduction of cysteine pairs that can form a disulfide bond thatstabilizes the C-terminal region of F1, and introduction of atransmembrane domain (e.g., for applications including a membrane-boundPreF antigen).

Disulfide Bonds.

One strategy for stabilizing the membrane proximal lobe of the F proteinis to introduce one or more cysteine substitutions that introduce adisulfide bond that that stabilizes the C-terminal portion of F₁ (forexample, for an application including a soluble PreF antigen). Such astrategy can be combined with any of the stabilization modificationsprovided herein, for example, those described in Example 2, such as a F₁protein with a S155C/S290C cysteine substitution. One strategy includesintroduction of two cysteine residues that are within a sufficientlyclose distance to form an inter-protomer disulfide bond that links theC-terminal region of the F₁ protein in the prefusion conformation. Aninter-protomer disulfide bond would be formed between adjacent protomerswithin the trimer, and thus would cross-link the three protomerstogether. Using the methods described above, several pairs of residuesof the RSV F protein were determined to be in close enough proximity inthe prefusion conformation, to form an inter-protomer disulfide bond ifcysteines were introduces at the corresponding residue pair positions.

Examples of cysteine substitutions that can be introduced to generate adisulfide bond that stabilizes the membrane proximal lobe includecysteine substitutions at residue pairs: 486 and 487; 486 and 487, witha P insertion between positions 486/487; 512 and 513; 493, with a Cinsertion between 329/330; 493 with a C insertion between 329/330, and Ginsertion between 492/493. Further, the length of the F₁ polypeptide canbe varied, depending on the position of the of the C-terminal cysteinepair. For example, the F₁ polypeptide can include positions 137-481,which eliminate the α10 helix from the F₁ polypeptide.

Examples of constructs containing modifications including cysteines atthese residue pairs, as well as additional description are listed inTable 17, below. Table 17 also lists a SEQ ID NO containing theindicated substitutions, and corresponding to a precursor F₀ constructalso including a signal peptide, F₂ polypeptide (positions 26-109),pep27 polypeptide (positions 27-136), F₁ polypeptide (with varyingpositions).

TABLE 17 Disulfide bonds to stabilize the membrane proximal lobe of Fprotein. F₁ SEQ ID Substitutions/insertion Description positions NOD486C/E487C; The D486C and E487C mutations allows inter-protomer 137-481304 S155C/S290C disulfide bond formation, the S155C/S290C mutationsstabilize the prefusion format, no Foldon or alpha-10 helix.S155C/S290C; The D486C and E487C mutant should allow inter-protomer137-481 305 D486C/E487C; P disulfide bond formation while theS155C/S290C mutations insertion between stabilize the prefusion format,no Foldon or alpha-10 helix. positions 486/487 N183C/N428C; The D486Cand E487C mutant allows inter-protomer 137-481 306 D486C/E487C; Gdisulfide bond formation; the 183C and 428C mutations insertion betweenstabilize prefusion format. No Foldon or alpha-10 sequence. 182/183N183C/K427G; C The D486C and E487C mutant allows inter-protomer 137-481307 insertion between disulfide bond formation; the 183C and 428Cmutations stabilize 247/428; the prefusion format. no Foldon sequence oralpha-10 sequence. D486C/E487C; P insertion between positions 486/487V402C/L141C; The 141C and 402C stabilize the prefusion form by locking 1-513 308 L512C/L513C down the fusion peptide. While the 512C and 513Ccreate an inter-protomer disulfide bond. no Foldon domain. S155C/S290C;The 141C and 402C stabilize the prefusion form by locking  1-513 309V402C/L141C down the fusion peptide in conjunction with the L512C/L513CS155C/S290C. While the 512C and 513C create an inter- protomer disulfidebond. no Foldon sequence S155C/S290C; Removal of the “Foldon” and thefacilitation of intermolecular 137-491 310 S493C; C insertion disulfidebond stabilization while the S155C/S290C mutations between 329/330stabilize the prefusion format S155C/S290C; Removal of the “Foldon” andthe facilitation of intermolecular 137-491 311 S493C; C insertiondisulfide bond stabilization while the S155C/S290C mutations between329/330; stabilize the prefusion format G insertion between 492/493

Transmembrane Domains.

Another strategy for stabilizing the membrane proximal lobe of the Fprotein is to include a transmembrane domain on the F₁ protein, forexample, for an application including a membrane anchored PreF antigen.For example, the presence of the transmembrane sequences is useful forexpression as a transmembrane protein for membrane vesicle preparation.The transmembrane domain can be linked to a F₁ protein containing any ofthe stabilizing mutations provided herein, for example, those describedin Example 2, such as a F₁ protein with a S155C/S290C cysteinesubstitution. Additionally, the transmembrane domain can be furtherlinked to a RSV F₁ cytosolic tail. Examples of precursor F₀ constructsincluding a signal peptide, F₂ polypeptide (positions 26-109), pep27polypeptide (positions 27-136), F₁ polypeptide (positions 137-513), aRSV transmembrane domain are provided as SEQ ID NOs: 323 (without acytosolic domain) and 324 (with a cytosolic domain).

Example 4 Single Chain PreF Antigens

This example illustrates recombinant RSV F proteins that lack the nativefurin cleavage sites, such that the F protein protomer is formed as asingle polypeptide chain, instead of a F₂/F₁ heterodimer.

Table 18 lists several single chain PreF antigens that include deletionof F positions 98-149, which removes the two furin cleavage sites, thepep27 polypeptide, and the fusion peptide. The remaining portions of theF₁ and F₂ polypeptides are joined by a linker Additionally, severalstrategies can be employed to stabilize the single chain constructs in aprefusion conformation, including use of the strategies described inexamples 2 and 3, above. Table 18 also lists a SEQ ID NO containing theindicated substitutions, and corresponding to a precursor F₀ constructalso including a signal peptide, F₂ polypeptide (positions 26-109),pep27 polypeptide (positions 27-136), F₁ polypeptide (with varyingpositions).

TABLE 18 Single chain PreF antigens SEQ F2/F₁ C-term ID SubstitutionsDiscussion Linker Stabilization NO S155C/S290C(A) The rationale for this construct  GSGNVGLGG Foldon 313 L373Ris to create a single chain RSV   (SEQ ID Δ98-149fusion molecule, remove the nucleus    NO: 356)localization signal, and the fusion  peptide while the S155C/S290C mutations stabilize the prefusion  format S155C/S290C Same as (A)GSGNWGLGG Foldon 314 L373R (SEQ ID Δ98-149 NO: 357) S155C/S290CSame as (A) GSGNIGLGG Foldon 315 L373R (SEQ ID Δ98-149 NO: 358)S155C/S290C Same as (A) GSGGNGIGLG Foldon 316 L373R G (SEQ ID Δ98-149NO: 359) S155C/S290C Same as (A) GSGGSGGSGG Foldon 317 L373R (SEQ IDΔ98-149 NO: 360) S155C/S290C Same as (A) GSGNVLGG Foldon 318 L373R(SEQ ID Δ98-149 NO: 361) S155C/S290C(B) The rationale for this construct  GSGNVGLGG D486C/ 319 L373Ris to create a single chain RSV   (SEQ ID E487C; P Δ98-149fusion molecule, remove the nucleus   NO: 362) insertionlocalization signal, and the fusion   betweenpeptide and also the alpha 10 helix   positionsand Foldon, while the S155C/S290C   486/487mutations stabilize the prefusion  format S155C/S290C/ Same as (B)GSGNVGLGG L512C/ 320 L373R (SEQ ID L513C Δ98-149 NO: 363) S155C/S290CSame as (A) GSGNIGLGG TM 322 L373R (SEQ ID Δ98-149 NO: 364) S155C/S290CThe rationale is to create a  GSGNIGLGG TM + 325 L373Rtransmembrane single chain RSV  (SEQ ID Cytoplasmic Δ98-149molecule requiring the cytoplasmic  NO: 365) domaintail to allow generation of a  virus-like particle which may be a viable immunogen while the S155C/S290C mutations stabilize the prefusion format

Example 5 RSV F Protein Stabilized with a Disulfide Bond and aTrimerization Domain

This example illustrates production of a RSV F protein stabilized with adisulfide bond and a trimerization domain.

As illustrated in FIG. 10, the serine residues at positions 155 and 290(indicated by arrows and red highlighting in the ribbon diagrams) areadjacent to each other in the prefusion conformation of RSV F protein,but not in the post fusion conformation of the RSV F protein. Further,the side chains of these residues are oriented towards one another.However, the side chains of the residues adjacent to serine 155, valine154 and lysine 156, are oriented away from the side chain of serine 290.In view of these findings, a recombinant RSV F protein was constructedwith S155C and S290C substitutions. It was expected that the cysteineresidues in this 155/290 construct would form a disulfide bond thatwould lock the recombinant RSV F protein in the prefusion conformation,but that incorporation of cysteines at positions 154 or 156 (instead ofposition 155) would fail to produce a stabilizing disulfide bond.

A nucleic acid molecule encoding a native RSV F₀ polypeptide was mutatedusing standard molecular biology techniques to encode the RSV F proteincalled RSVF(+)FdTHS S155C, S290C, and set forth as SEQ ID NO: 185:MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSHHHHHHSAWSHPQFEK (SEQ ID NO: 185).

RSVF(+)FdTHS S155C, S290C includes a signal peptide (residues 1-25), F₂polypeptide (residues 26-109), Pep27 polypeptide (residues (27-136), F₁polypeptide (residues 137-513), Foldon domain (residues 514-544), and athrombin cleavage site (LVPRGS) and purification tags (his-tag (HHHHHH)and Strep Tag II (SAWSHPQFEK)). Control constructs were also generatedwith V154C or K156C substitutions instead of the S155C substitution.When expressed in cells, RSVF(+)FdTHS S155C, S290C was processed andexpressed as a stable and soluble RSV F protein; however, the controlconstructs with 154/290 or 156/290 substitutions failed to express(likely because they failed to fold in a soluble conformation) (see FIG.10).

The RSVF(+)FdTHS S155C, S290C construct was purified and tested forantibody binding to the prefusion specific antibodies AM22 and D25, aswell as 131-2a antibody (which binds antigenic site I, present on pre-and post-fusion RSV F conformations), motavizumab and palivizumab (whichbind antigenic site II, present on pre- and post-fusion RSV Fconformations), and 101F antibody (which binds antigenic site IV,present on pre- and post-fusion RSV F conformations). As shown in FIG.11 (left graph), all of these antibodies specifically bound to thepurified RSVF(+)FdTHS S155C, S290C construct, indicating thatRSVF(+)FdTHS S155C, S290C maintains a prefusion conformation. Theresults further indicate that this construct maintains antigenic sitesI, II and IV, common to the pre- and post-fusion RSV F conformations.

To demonstrate that purified RSVF(+)FdTHS S155C, S290C is in a trimericconformation, this construct was passed over a size-exclusionchromatography column. As shown in FIG. 11 (right graphs) a preparationof purified RSVF(+)FdTHS S155C, S290C eluted in a single peakcorresponding to the molecular weight of the trimeric F protein. Incontrast, a preparation of a control construct lacking the S155C andS290C substitutions, which is not expected to be stabilized in theprefusion conformation, eluted in multiple peaks, indicating thepresence of rosettes of triggered F protein and aggregates, indicatingthat this control construct is not stable in a homogeneous prefusionconformation.

To further confirm that the RSVF(+)FdTHS S155C, S290C construct isstabilized in a prefusion conformation, electron microscopy studies wereperformed (FIG. 12) and demonstrate that RSVF(+)FdTHS S155C, S290C formhomogeneous population of structures with a shape similar to that of theprefusion conformation of RSV F, and significantly different from thatof the postfusion F protein (right image, from Martin et al., J. Gen.Virol., 2006).

Crystallography studies were performed to demonstrate that purifiedRSVF(+)FdTHS S155C, S290C is homogeneous in solution. Formation ofcrystals in aqueous solution is a stringent test for the homogeneity ofa protein in solution. FIG. 15 shows pictures of the crystals formed bypurified RSVF(+)FdTHS S155C, S290C in aqueous buffer containing 0.2 Mlithium sulfate, 1.64 M Na/K tartrate and 0.1 M CHES, at pH 9.5. Theformation of RSVF(+)FdTHS S155C, S290C crystals in aqueous bufferdemonstrates that this protein is substantially homogeneous in solution.

Example 6 Induction of a Neutralizing Immune Response Using a PreFAntigen

This example illustrates use of a PreF antigen to elicit a RSVneutralizing immune response in a subject.

Eight week old pathogen-free CB6F1/J mice (Jackson Labs) were dividedinto 5 groups of 10 each, and immunized with the following regimens:

1) live RSV A2 (RSV) at 5×10⁶ pfu intranasally;2) formalin-inactivated alum-precipitated RSV(FI-RSV) intramuscularly(IM);3) stabilized prefusion RSV F (RSVF(+)FdTHS S155C, S290C; prefusion F)20 μg in polyI:C 50 μg IM;4) postfusion RSV F trimer (postfusion RSV) 20 μg in polyI:C 50 μg IM;and5) recomb. adenovirus serotype 5 vector expressing wild-type RSV F(rAd5-F) 10⁹ particle units (PU) IM.

Group 1 (live RSV) was infected once at time 0, and all other groupswere immunized at 0 and 3 weeks. Serum was obtained at week 5, two weeksafter the 2^(nd) IM injection or five weeks post RSV infection.Neutralizing activity was determined by the following method: Sera weredistributed as four-fold dilutions from 1:10 to 1:40960, mixed with anequal volume of recombinant mKate-RSV expressing prototypic F genes fromeither strain A2 (subtype A) or 18537 (subtype B) and the Katushkafluorescent protein, and incubated at 37° C. for one hour. Next, 50 μlof each serum dilution/virus mixture was added to HEp-2 cells that hadbeen seeded at a density of 1.5×10⁴ in 30 μl MEM (minimal essentialmedium) in each well of 384-well black optical bottom plates, andincubated for 20-22 hours before spectrophotometric analysis at Ex 588nm and Em 635 nm (SpectraMax Paradigm, Molecular Devices, Sunnyvale,Calif. 94089). The IC50 for each sample was calculated by curve fittingand non-linear regression using GraphPad Prism (GraphPad Software Inc.,San Diego Calif.). P values were determined by Student's T-test. Theabove method for measuring RSV neutralization was performedsubstantially as described previously (see, e.g., Chen et al. J.Immunol. Methods., 362:180-184, 2010, incorporated by reference herein),except that the readout was by a fluorescent plate-reader instead offlow cytometry.

Using this assay, generally antibody responses above ˜100 EC50 would beconsidered to be protective. As shown in FIGS. 13 and 14, miceadministered an RSV F protein stabilized in a prefusion conformation(RSV F (RSVF(+)FdTHS S155C, S290C) produced a neutralizing immuneresponse to RSV A ˜15-fold greater than that produced by miceadministered a RSV F protein in a postfusion conformation, and aresponse to RSV B ˜5-fold greater than that produced by miceadministered a RSV F protein in a postfusion conformation. FIG. 13 showsthe results after 5 weeks post-initial immunization, and FIG. 14 showsresults after 7 weeks post immunization. The mean elicited IC50 valuesare also shown in FIGS. 13 and 14. The difference in neutralizationbetween RSV A and B subgroups is not surprising as the RSVF(+)FdTHSS155C, S290C construct is derived from a F protein from an RSV Asubgroup. It is expected that immunization with a correspondingconstruct derived from a RSV B strain would generate neutralizing seramore specific for RSV B. The results show that immunization with a RSV Fprotein stabilized in a prefusion conformation produces a protectiveimmune response to RSV.

Example 7 Treatment of Subjects with the Disclosed Vaccines

This example describes methods that can be used to treat a subject thathas or is at risk of having an infection from RSV by administration ofone or more of the disclosed PreF antigens. In particular examples, themethod includes screening a subject having, thought to have, or at riskof having (for example due to impaired immunity, physiological status,or exposure to RSV) an RSV infection. Subjects of an unknown infectionstatus can be examined to determine if they have an infection, forexample using serological tests, physical examination, enzyme-linkedimmunosorbent assay (ELISA), radiological screening or other diagnostictechnique known to those of ordinary skill in the art. In some examples,a subject is selected that has an RSV infection or is at risk ofacquiring an RSV infection. Subjects found to (or known to) have an RSVinfection and thereby treatable by administration of the disclosed PreFantigens are selected to receive the PreF antigens. Subjects may also beselected who are at risk of developing an influenza infection forexample, the elderly, the immunocompromised and the very young, such asinfants.

Subjects selected for treatment can be administered a therapeutic amountof disclosed PreF antigens. An immunogenic composition including thePreF antigen can be administered at doses of 1 μg/kg body weight toabout 1 mg/kg body weight per dose, such as 1 μg/kg body weight-100μg/kg body weight per dose, 100 μg/kg body weight-500 μg/kg body weightper dose, or 500 μg/kg body weight-1000 μg/kg body weight per dose oreven greater. However, the particular dose can be determined by askilled clinician. The immunogenic composition can be administered inseveral doses, for example continuously, daily, weekly, or monthly.

The mode of administration can be any used in the art, such as nasaladministration. The amount of agent administered to the subject can bedetermined by a clinician, and may depend on the particular subjecttreated. Specific exemplary amounts are provided herein (but thedisclosure is not limited to such doses).

It will be apparent that the precise details of the methods orcompositions described may be varied or modified without departing fromthe spirit of the described embodiments. We claim all such modificationsand variations that fall within the scope and spirit of the claimsbelow.

We claim:
 1. An isolated immunogen, comprising: a recombinant RSV Fprotein or fragment thereof comprising at least one amino acidsubstitution compared to a native RSV F protein that stabilizes therecombinant RSV F protein in a prefusion conformation that specificallybinds to a RSV F prefusion specific antibody, wherein: the immunogenspecifically binds to the antibody after incubation at 20° C. inphosphate buffered saline at physiological pH for at least 24 hours inthe absence of the antibody, and wherein the antibody does notspecifically bind to a RSV F protein in a post-fusion conformation. 2.The immunogen of claim 1, wherein the immunogen is at least 90%homogeneous and specifically binds to the prefusion antibody after theincubation at 20° C. in phosphate buffered saline at physiological pHfor at least 24 hours in the absence of the antibody.
 3. The immunogenof claim 1, wherein the recombinant RSV F protein comprises an antigenicsite Ø comprising residues 62-69 and 196-209 of SEQ ID NO: 370, thatspecifically binds a D25 antibody, or an AM22 antibody, or both.
 4. Theimmunogen of claim 1, wherein the amino acid sequence of the recombinantRSV F protein is at least 90% identical to the amino acid sequence ofthe native RSV F protein.
 5. The immunogen of claim 1, wherein thenative RSV F protein is from a human subtype A or subtype B, or bovineRSV.
 6. The immunogen of claim 1, wherein the recombinant RSV F protein(a) comprises an F₁ polypeptide; (b) comprises an F₂ polypeptide; (c)does not comprise a pep27 polypeptide or portion thereof; or (d) acombination of two or more of (a)-(c).
 7. The immunogen of claim 6,wherein the F₂ and F₁ polypeptides comprise RSV F positions 26-109 and137-513, respectively.
 8. The immunogen of claim 1, wherein therecombinant RSV F protein is a single chain RSV F protein.
 9. Theimmunogen of claim 1, wherein the recombinant RSV F protein isstabilized in the prefusion conformation by a cavity-filling amino acidsubstitution, a non-natural disulfide bond between a pair of cysteines,or both.
 10. The immunogen of claim 9, wherein the pair of cysteinescomprises a first cysteine and a second cysteine, each comprising a Cαcarbon and a Cβ carbon, and wherein: (A) the first cysteine isintroduced by amino acid substitution onto one of RSV F positions137-216 or 461-513, and the second cysteine is introduced by amino acidsubstitution onto one of RSV F positions 26-61, 77-97, or 271-460; and(B) the Cα carbon of the position of the first cysteine is from 2.0-8.0angstroms from the Cα carbon of the position of the second cysteine,and/or the Cβ carbon of the position of the first cysteine is from2.0-5.5 angstroms from the Cβ carbon of the position of the secondcysteine, in the three-dimensional structure set forth by the structuralcoordinates provided in Table
 1. 11. The immunogen of claim 9, whereinthe pair of cysteines comprises a first cysteine and a second cysteine,each comprising a Cα carbon and a Cβ carbon, and wherein: (A) the firstcysteine and the second cysteine are introduced by amino acidsubstitution onto RSV F positions 137-216 or RSV F positions 461-513; orthe first cysteine is introduced by amino acid substitution onto RSV Fpositions 137-216, and the second cysteine is introduced by amino acidsubstitution onto RSV F positions 461-513; and (B) the Cα carbon of theposition of the first cysteine is from 2.0-8.0 angstroms from the Cαcarbon of the position of the second cysteine, and/or the Cβ carbon ofthe position of the first cysteine is from 2.0-5.5 angstroms from the Cβcarbon of the position of the second cysteine, in the three-dimensionalstructure set forth by the structural coordinates provided in Table 1.12. The immunogen of claim 9, wherein the non-natural disulfide bondcomprises an intra-protomer disulfide bond between RSV F positions 155and 290; 151 and 288; 137 and 337; 397 and 487; 138 and 353; 60 and 194;33 and 469; 54 and 154; 59 and 192; or; an inter-protomer disulfide bondbetween RSV F positions 400 and 489; 144 and 406; 153 and 461; 149 and458; 143 and 404; 399 and 494; 146 and 407; 402 and 141; 183 and 428,and the recombinant RSV F protein comprises a G insertion betweenpositions 182/183; 183 and 428, and the recombinant RSV F proteincomprises a C insertion between positions 427/428; 145 and 460, and therecombinant RSV F protein comprises a AA insertion between positions146/147; 183 and 423, and the recombinant RSV F protein comprises a AAAinsertion between positions 182/183; or 330 and 430, and the recombinantRSV F protein comprises a CAA insertion between positions 329/330; theintra-protomer disulfide bond between RSV F positions 155 and 290, andwherein the recombinant RSV F protein further comprises a non-naturaldisulfide bond between RSV F positions; 141 and 402; 146 and 460, and aG insertion between positions 460/461; 330 and 493, and a C insertionbetween positions 329/330; 183 and 428, and a G insertion betweenpositions 182/183; or 183 and 428, and a C insertion between positions427/428.
 13. The immunogen of claim 12, wherein the recombinant RSV Fprotein comprises: the intra-protomer disulfide bond, and one or more ofthe following sets of substitutions: S155C and S290C; G151C and I288C;F137C and T337C; T397C and E487C; L138C and P353C; E60C and D194C; Y33Cand V469C; T54C and V154C; I59C and V192C; or the inter-protomerdisulfide bond, and one or more of the following sets of substitutions:T400C and D489C; V144C and V406C; A153C and K461C; A149C and Y458C;G143C and S404C; K399C and Q494C; S146C and I407C; or V402C and L141C;S155C, S290C, L141C, and V402C; S155C, S290C; N183C and N428C, and a Ginsertion between positions 182/183; N183C and N427G, and a C insertionbetween positions 427/428; S145C and 460C; and an AA insertion betweenpositions 146/147; N183C and K423C, and an AAA insertion betweenpositions 182/183; A329C and S430C, and a CAA insertion betweenpositions 329/330; or the intra-protomer disulfide bond between RSV Fpositions 155 and 290 and the additional non-natural disulfide bond,S155C and S290C substitutions, and one or more of the following sets ofamino acid substitutions: S146C, and N460C, and a G insertion betweenpositions 460/461; N183C, and N428C; and a G insertion between positions182/183; or N183C, and N427G; and a C insertion between positions427/428.
 14. The immunogen of claim 13, wherein the recombinant RSV Fprotein comprises an F₁ polypeptide comprising the amino acid sequenceset forth as residues 137-513 of one of SEQ ID NOs: 185, 189, 201, 202,205, 207, 209, 213, 244, 245, 247, 257-262, 264-275, 277-282, 284,296-299, 302, 303, 338-340; or an F₂ polypeptide and an F₁ polypeptidecomprising the amino acid sequences set forth as residues 26-109 and137-513, respectively, of one of SEQ ID NOs: 190, 211, 212, 243, 246,263, 276, 283,
 285. 15. The immunogen of claim 9, wherein the cavityfilling amino acid substitution comprises cavity filling amino acidsubstitutions at RSV F positions 190 and 207, 207 and 220, 296 and 190,220 and 153, 203, 83 and 260, 58 and 298, 87 and 90, or a combinationthereof.
 16. The immunogen of claim 15, wherein the recombinant RSV Fprotein comprises one or more cavity-filling amino acid substitutionscomprising 190F and 207L substitutions, 207L and 220L substitutions,296F and 190F substitutions, 220L and 153W substitutions, a 203Wsubstitution, 83W and 260W substitutions, 58W and 298L substitutions,87F and 90L substitutions, or a combination thereof.
 17. The immunogenof claim 16, wherein the cavity-filling amino acid substitutionscomprise S190F and V207L substitutions, V207L and V220L substitutions,V296F and S190F substitutions, V220L and A153W substitutions, a L203Wsubstitution, L83W and L260W substitutions, T58W and A298Lsubstitutions, K87F and V90L substitutions, or a combination thereof.18. The immunogen of claim 17, wherein the recombinant RSV F proteincomprises an F₁ polypeptide comprising positions 137-513 of one of SEQID NOs: 191, 193, 196, 197, or 248; an F₂ polypeptide and an F₁polypeptide comprising residues 26-109 and 137-513, respectively, of oneof SEQ ID NOs: 192, 195 or
 194. 19. The immunogen of claim 1, whereinthe recombinant RSV F protein further comprises a non-natural disulfidebond to stabilize the membrane proximal domain of the RSV F protein,wherein the non-natural disulfide bond to stabilize the membraneproximal domain is between RSV positions 512 and 513; 486 and 487; 486and 487, and the recombinant RSV F protein comprises a P insertionbetween positions 486/487; 330 and 493, and the recombinant RSV Fprotein comprises a C insertion between positions 329/330; or 330 and493, and the recombinant RSV F protein comprises a C insertion betweenpositions 329/330 and a G insertion between positions 492/493, whereinthe amino acid positions correspond to positions of the reference F₀polypeptide set forth as SEQ ID NO:
 124. 20. The immunogen of claim 1,wherein the C-terminus of recombinant RSV F protein is linked to atrimerization domain.
 21. The immunogen of claim 20, wherein thetrimerization domain is a Foldon domain.
 22. The immunogen of claim 21,wherein the recombinant RSV F protein comprises the amino acid sequencesset forth as positions 26-109 and 137-544 of any one of SEQ ID NOs: 185,189-197, 201-202, 205, 207, 209, 211-213, 243-248, 257-285, 296-299, or302-303.
 23. The immunogen of claim 1, wherein the C-terminus ofrecombinant RSV F protein is linked to a ferritin domain, an encapsulindomain, a Sulfur Oxygenase Reductase (SOR) domain, a lumazine synthasedomain, or a pyruvate dehydrogenase domain.
 24. The immunogen of claim1, wherein the C-terminus of recombinant RSV F protein is linked to atransmembrane domain.
 25. The immunogen of claim 1, wherein (a) theprefusion specific antibody specifically binds to the immunogen afterthe immunogen is incubated at 20 degrees Celsius in phosphate bufferedsaline at pH 7.4 for at least 24 hours in the absence of the prefusionspecific antibody; (b) at least 90% of the recombinant RSV F proteins inthe homogeneous population are stabilized in the prefusion conformation.(c) at least 90% of the recombinant RSV F proteins in the homogeneouspopulation are specifically bound by a RSV F protein prefusion specificantibody; or (d) a combination of two or more of (a)-(c).
 26. Avirus-like particle comprising the immunogen of claim
 1. 27. A proteinnanoparticle comprising the immunogen of claim
 1. 28. The proteinnanoparticle of claim 26, wherein the protein nanoparticle is a ferritinnanoparticle, an encapsulin nanoparticle, a Sulfur Oxygenase Reductase(SOR) nanoparticle, a lumazine synthase nanoparticle, or a pyruvatedehydrogenase nanoparticle.
 29. A nucleic acid molecule encoding theimmunogen of claim
 1. 30. A vector comprising the nucleic acid moleculeof claim
 29. 31. An isolated host cell comprising the vector of claim30.
 32. An immunogenic composition comprising an effective amount of theimmunogen of claim 1, and a pharmaceutically acceptable carrier.
 33. Theimmunogenic composition of claim 32, further comprising an adjuvant. 34.The immunogenic composition of claim 33, wherein the adjuvant promotes aTh1 immune response.
 35. A method for generating an immune response toRSV F protein in a subject, comprising administering an effective amountof the immunogenic composition of claim 32 to the subject to generatethe immune response.
 36. The method of claim 35, wherein the immuneresponse comprises a Th1 immune response.
 37. The method of claim 35,comprising a prime-boost administration of the immunogenic composition.38. The method of claim 35, wherein the subject is a human subject orcattle or sheep.
 39. The method of claim 35, wherein the subject is lessthan one year old and immunogenic composition comprises an AS01adjuvant; or more than 65 years old and the immunogenic compositioncomprises an AS04 adjuvant.
 40. The method of claim 35, wherein thesubject is pregnant.
 41. The method of claim 40, wherein the immunogeniccomposition comprises an adjuvant and wherein the adjuvant comprisesAlum.
 42. A method for treating or preventing a RSV infection in asubject, comprising administering to the subject a therapeuticallyeffective amount of the immunogenic composition of claim 32, therebytreating or preventing RSV infection in the subject.
 43. A kitcomprising the immunogen of claim 1 and instructions for using the kit.44. An epitope-scaffold protein, comprising a heterologous scaffoldprotein linked to a polypeptide comprising a RSV F protein prefusionepitope, wherein monoclonal antibody D25 or AM22 specifically binds tothe epitope-scaffold protein.
 45. The epitope scaffold protein of claim43, comprising RSV F₂ positions 62-69, and RSV F₁ positions 196-209,wherein the positions correspond to positions of the reference F₀polypeptide set forth as SEQ ID NO:
 124. 46. A recombinant RSV F proteincomprising cysteine residues at positions 155 and 290, wherein the RSV Fprotein induces an immune response to RSV F protein in a subject. 47.The recombinant RSV F protein of claim 45, wherein the recombinant RSV Fprotein is a single chain RSV F protein.